Japanese pharmaceutical companies have struggled to find a way into the global marketplace. According to Tadayoshi Kawasaki, Nihon Millipore KK, with government support, it won\'t be long before Japan makes its presence felt worldwide in the development and manufacturing of biopharmaceuticals.
In 2002, the Japanese Ministry of Education, Culture, Sports, Science and Technology (MECSST) launched its second Basic Programme for Science and Technology for 2003-07. The previous programme focused on promoting basic research and set out to improve Japan's ability to compete at a world level in science and technology. The new programme focuses on life sciences, information technology, environmental science, nanotechnology and materials, as well as on advanced areas of science and technology, such as bioinformatics, system biology and nano-biology. A third programme has already been discussed and will be announced later in 2006.
Japan's Ministry of Economy, Trade and Industry has a similar vision for the pharmaceutical industry, in four different business areas:
Although Japan has not yet produced a fully global pharma company, during the last couple of years, Japan's top five pharmas have increased their business outside Japan.
Much of Japan's traditional food and beverages have been produced through fermentation technology - sake and soy sauce, for example, are produced using yeast and malt enzymes. At the same time, Japanese scientists have a history of producing important chemicals: Dr Umetaro Suzuki discovered and developed Vitamin B1, and Dr Kikunae Ikeda discovered sodium glutamate and amino acids - chemicals used as intermediates for pharmaceuticals. As a result, Japanese companies have been well placed to exploit fermentation technology in the production of amino acids, vitamins and antibiotics.
Today, Japanese companies are focusing on developing innovative new products based on fermentation technology, for example, microelectronics. Fermentation technologies are also being applied in the design and manufacture of microfluidics and micro-arrays in biotechnology and bioscience. Such micro-manipulation also opens up new opportunities in gene manipulation, genomics and proteomics, and more recently, glycotechnologies. These technologies will also be used in drug screening and research.
In 2004, Japan's biotechnology market had an estimated value of $15.2bn, while the biotech market was worth $10.9bn.
Since the end of the Human Genome Project, researchers moved to proteome and post-translational modifications. The Japanese government h4ly supports the development of technology for this area, and has introduced a principle of competition for research grants, forcing academia to compete on global terms and to strengthen its R&D.
Compared with the pharmaceutical industry, biopharma has been slow to move into new areas. Since 1985, the compound annual growth rate has increased by only 2.5 per cent, while European and US pharmas have grown by over 10 per cent, some of this through mergers and acquisitions. During the last couple of years, some Japanese companies have started to expand through collaborations, acquisitions and mergers.
In 2002, Chugai Pharmaceuticals Inc, Japan's top biopharma company, was acquired by Roche, which accelerated the development and manufacturing of protein pharmaceuticals. In 2003, Taisho and Kowa moved to OTC, while Teikoku Hormon and Grelan merged to become Aska Pharmaceuticals, a contract manufacturing business. In 2005, three large mergers were announced. Daiich Pharma was acquired by Sankyo and with the purchase of Suntory Pharma became Daiichi-Sankyo Pharmaceuticals. Yamanouch and Fujisawa merged and split their businesses to become Astellas Pharma, a pharmaceuticals manufacturing firm, and Zepharma, an OTC and generics company. Dainippon Pharma was acquired by Sumitomo Pharmaceuticals.
The generic pharmaceutical sector has not grown much in Japan compared with the rest of the world, mainly because people prefer to use original, branded products. However, the Ministry of Health, Labour and Welfare, concerned about increasing expenditure on drugs, hopes to change this perception and is encouraging the use of generics.
Japan's biopharma market has relatively few monoclonal antibodies. The top five biopharmaceuticals marketed in Japan are: EPO, the growth hormone, CSF, insulin and interferon. However, there have been some advances in monoclonal antibody therapeutics in Japan, which despite being a small market, is growing.
Japanese companies that are active in biopharma are engaged in a number of interesting developments:
The production of recombinant human serum albumin (rHSA) is potentially one of the biggest healthcare applications for any recombinant protein, with world demand at 500 tonnes. The Bipha Corporation, which is 51 per cent owned by Mitsubishi Pharma and 49 per cent owned by NIPRO, can produce 12.5 tonnes of pure rHSA each year. It has built a new facility in Chitose, which is expected to produce over 40 tonnes per year, when the second stage of construction is finished.
Bipha uses an interesting expression system, Pichia yeast, which can be fed on methanol. A very high expression level has been achieved.
STREAMLINE™ vs Conventional Technology
To produce such a huge amount of protein as pharmaceuticals, several key breakthroughs were required (see Figure 17). The first breakthrough was at the expression stage, where market and economic requirements needed to be met. The second occurred at the downstream stage. Bipha has a huge bioreactor for yeast cell culture, which processes huge volumes of fermentation broth. In the old, conventional process, three to four processes were used for clarification, and then the targeted component was captured using ion exchange chromatography. However, for this particular clarification and capturing process, an expanded bed adsorption (EBA) technology was used - Streamline - which reduced the process operations to one. Overall, the use of EBA reduced time and cost, while dramatically increasing productivity.
Chugai Pharmaceuticals Inc, 51 per cent owned by Roche, took up the challenge of manufacturing a large amount of Mab. The target substance is a humanised anti-human IL-6 (interleukin-6) receptor Mab, tocilizumab (genetical recombination) injection, trade name ACTEMRA(r), which was co-developed with Osaka University. It was approved for Castleman's disease during 2005, but it is now being developed for use in cancer, inflammation and chronic diseases such as rheumatoid arthritis. It is the first Mab therapeutic originally developed in Japan.
To manufacture the Mab, Chugai constructed a new facility at its Utsunomiya plant. The first stage of construction began in February 2001, with two 10,000l bioreactors for recombinant CHO cell culture. Chugai has recently added a further six 10,000l bioreactors. With such large-scale bulk manufacturing, clarification and cell removal is a key process. If this process takes too long, the product can be damaged by the proteases. A process to remove cells with minimum damage was required because, if cells are disrupted, a complex cytoplasimic component is produced, such as enzymes and DNA/RNA, which would complicate the downstream process.
There are some major difficulties with Mab clarification. The most important one is the need to reduce the volume of water required in the short process time. The second relates to the complexity of the feed. Recently, a large amount of hydrolysate was used for high-density animal cell culture, causing difficulties in the downstream process.
In the clarification process for animal cell culture, continuous centrifugation is commonly used. Other techniques are tangential flow filtration (TFF) and normal flow filtration (NFF), or a combination of these techniques, which is required for a very high cell density culture, of over 107 cells/ml, for example.
Optimising conditions for the combination of centrifugation and NFF using a depth-type filter is a simple process using a pressure resistance of filtration and the turbidity measurement for the filtrate. If the centrifugation process is well optimised, the depth filter filtration process is efficient and it is unlikely that there will be a clogging problem.
However, in general, depth-type filtration is not a clean process, because the filter is placed in a large dome that, after the processing of a culture broth, must be removed and cleaned. To avoid such a messy process, a cartridge-type depth filter was launched, the Mill-Pod, or Millistak Pod, resulting in a clean clarification process.
In general, centrifugation and depth-type filtration are used for small- to medium-scale processes, up to 1000l or 2000l animal cell culture. However, for the manufacture of Mab, bioreactors of over 10,000l are used. In such large-volume clarification, it is important to remove cells as quickly as possible to avoid degradation.
One solution is to use the tangential flow micro filtration (MF-TFF) technique. For most cases of CHO cell removal, it is used with either 0.45 or 0.65 micron pore membranes and works efficiently.
Another biopharma challenge is the use of the silkworm as an expression host, and baculovirus as a vector. This is one well-established technique not commonly used for recombinant protein manufacturing. However, the silkworm can achieve a high level of protein expression, and there are many small projects that use the insect or insect cell to express recombinant proteins. However, these are mostly at the research stage.
Toray Industry Inc uses silkworms to produce the recombinant protein. It has developed and launched recombinant interferon as a veterinary medicine for dogs and cats. It has also developed its own expression system by using baculovirus in the silkworm for transfecting and expressing interferon in the body. Downstream processes have been developed to purify the protein from the silkworm's body fluid. So far, the recombinant protein has only been used for the treatment of animals, not humans.
The technology is promising, but there are some issues that need to be resolved if the process is to be used for humans. One is that a post-translational modification is not identical to that of humans, in particular the glycosylation form. Another concern relates to viruses. It is unclear if mammalian viruses would infect the insect cell lines of interest.
Transgenics as Expression Host
So far, many different transgenics have been developed and used in the manufacture of biopharmaceuticals - several animals and plant are of interest. Advanced projects in this area are undergoing clinical studies.
GTC Biotherapeutics, previously known as Genzyme Transgenics, has used a transgenic animal to produce milk for biopharmaceutical manufacturing. The desired DNA is injected with a vector into fertilised embryos and transferred into the recipient female. The offspring are tested for the transgene, and carriers then rebred to develop the milk production herd.
Transchromosone Technology: How transgenic animals
are used in biopharmaceutical manufacturing
Kirin Brewery is one of Japan's top breweries. It is also recognised as an advanced biopharmaceutical company, producing recombinant erythropoietin (EPO) and granulocyte colony-stimulating factor (GCSF) at its own facility in collaboration with Amgen. Kirin has developed an advanced trans-chromosome technology (see Figure 36), making it possible to transfect the full length of the human immunoglobulin gene.
The company has also bred trans-chromosome mice with a fully human immunoglobulin gene, allowing the development of a fully human monoclonal antibody. Recently, it has bred a trans-chromosome cow with a fully human immunoglobulin gene. It has also demonstrated the possibility of producing polychronal immunoglobulin.
Many Japanese pharmaceutical companies have struggled to find their way into global markets. Traditionally, pharmaceutical companies did not invest much in the development of biopharmaceuticals, choosing to remain in chemical and small-molecule pharmaceuticals. However, Japan's pharmaceutical industry is now developing several different business areas, including biopharmaceuticals.
The Japanese government h4ly supports biotechnology and nano-technology, and as a result, these are being applied in the development of biopharmaceuticals. Some companies are also developing their own advanced technology for the development and manufacturing of biopharmaceuticals.