Susan Aldridge

Arid development pipelines and the maturation of therapeutic antibody products in recent years have caused many companies operating in this sector to look for new ways to sustain growth. Although research continues in follow-on biologics and novel methods of antibody drug delivery, it will be some time before these innovations translate into revenues. As such, partnerships are the perfect solution for many firms seeking to secure a healthy future. 

One example is the recent announcement of a merger between antibody discovery company Affitech (Norway) and therapeutic antibody manufacturer Pharmexa (Denmark). Affitech's phagemid antibody display technology, licensed from the German Cancer Research Centre, makes fully human antibodies against some interesting new targets in cancer and other disease areas. The deal with Pharmexa will provide the company with the development infrastructure to begin commercializing these products. Affitech A/S, as the new company will be known, will focus on the discovery and development of antibodies against challenging targets, such as G-protein coupled receptors (GPCRs) and cancer stem cell antigens, as well as looking for new and improved versions of existing therapeutic antibody products.  

An inter-biotech merger is often the less preferred strategy for those seeking suitable partners as many biotechs prefer to link up with large pharmaceutical companies that can offer greater sales, marketing power and experience. Examples of deals between antibody manufacturing biotech firms and Big Pharma include the late 2006 acquisition of UK-based Domantis by GlaxoSmithKline (GSK). Widely regarded as the pioneer of 'next generation' therapeutic antibodies, and founded as a spin-off from the work of Sir Greg Winter of the Laboratory of Molecular Biology in Cambridge (UK), Domantis was made part of GSK's Centre of Excellence for Drug Discovery. Winter's first breakthrough, in 1986, came when he made the shift from the original mouse-derived monoclonal antibodies, which were not really suitable for clinical application, to humanized versions, which are. However, these large molecules could only be administered by injection or infusion; the research that led to the founding of Domantis involved tailoring the molecules to produce the smallest functional therapeutic binding unit. Through Domantis's research efforts, these domain antibodies can now be delivered by inhalation or topically and even, potentially, orally.  

At about the same time, Cambridge Antibody Technology (CAT) (as with Domantis; also a Medical Research Council spin-off) was bought by AstraZeneca. CAT is famous for developing Humira; the first fully human antibody to be commercialized. This company is now known as MedImmune Cambridge.  

Big Pharma's interest in acquiring therapeutic antibodies — either by buying up companies or in-licensing candidate molecules — has grown as the sector has come of age during the last 10 years. In fact, some antibodies have now reached blockbuster status. US-based biotech firm Genentech, in particular, boasts several such blockbuster-level antibodies in the form of Her-2 receptor antagonist Herceptin (trastuzumab), anti-angiogenesis agents Avastin (bevacizumab) and Lucentis (ranibizumab), and B-cell therapy Rituxan (rituximab), which are indicated for various cancers, retinal disease and inflammatory conditions. Meanwhile, US firms Centocor and Abbott have also enjoyed success in this area with their antibodies Remicade (rituximab; indicated for the treatment of leukaemia) and Humira (adalimumab; indicated for the treatment of rheumatoid arthritis), respectively. 

Although antibody R&D has already yielded some revolutionizing treatments for oncological, inflammatory and ocular conditions, sales forecasts for currently marketed and pipeline therapeutics are still strong. According to Mario Brkulj, Senior PR Manager at antibody manufacturer MorphoSys (Germany), the therapeutic antibody market was worth $26 billion in 2008 and this is likely to double by 2013. 

Thanks to developments in manufacturing methods and cell lines used to develop antibodies, many problems that once plagued antibody manufacturers, such as antibody characterization, manufacturing capacity and purification, have largely been resolved. The products themselves are more advanced and can now be successfully developed from fully human, rather than animal, cell lines; a requirement that is increasingly being demanded by regulatory agencies. These next generation antibodies have improved clinical safety and better physicochemical and clinical properties than the 'old' monoclonals. Expression systems have improved so manufacturing yields are up and, with the advent of new chromatography systems and the introduction of disposables for speed and quality, there have been many improvements in downstream processing too. The regulators now seem, on the whole, more comfortable with this class of therapeutics. 

This class of drugs is, however, not without its horror stories. One such case to hit the headlines was in March 2006 when Phase I testing of an experimental biologic, TGN 1412, caused the death of three young volunteers in the UK.  

Nonetheless, research continues and, in the wake of the Human Genome Project, which provided many new genes for researchers to study, increasing numbers of drug targets are being discovered and, more importantly, validated in disease areas such as oncology, osteoporosis and inflammation. Couple this with new antibody discovery technologies and there is the potential for many more blockbusters. To name just one example, Affitech's compound R84, which is being codeveloped with Peregrine Pharmaceuticals (CA, USA), is a powerful inhibitor of angiogenesis that could, one day, rival Genentech's cash cow Avastin.  

Long-standing German antibody manufacturer MorphoSys also has a number of promising candidates on the horizon. Unlike many biotechs, MorphoSys has retained its independence through an established deal with Novartis, and various agreements with other large pharma companies, such as Merck and Pfizer, have secured a healthy flow of cash for continued research and innovation. Using its patented HuCAL (Human Combinatorial Antibody Library) technology — an automatic and rapid tool for the production of optimized and fully human antibodies — MorphoSys has selected two therapeutic antibody candidates for development. Now in Phase II clinical trials, MOR103 is being developed for the treatment of rheumatoid arthritis, whilst MOR202 targets multiple myeloma and certain leukaemias.  

Elsewhere, a Belgian firm has focused on the production of small, compact antibodies, which it calls Nanobodies. Ablynx was founded following the discovery of therapeutic antibodies from camels, llamas and related species by a team of researchers at the University of Brussels. Chief Business Officer of Ablynx, Eva-Lott Allan, describes their exciting antibody candidates as "large, small molecules" that have properties closer to conventional pharmaceuticals than biologic drugs. Derived from immunized llamas, nanobodies are only one-tenth of the size of a conventional antibody and are better at penetrating tissue, therefore, promising greater clinical efficacy. Additionally, unlike larger protein molecules, they are relatively resistant to degradation and offer more possibilities for drug delivery (therefore, Ablynx has recently been working on pulmonary formulations). "The key feature of nanobodies is that they are stable so they can be stored and, from a formulation point of view, they can be encapsulated and lyophilized," explains Allan. She also points out that one of the big advantages of nanobodies is that they can be manufactured in a microbial host, which is simpler and cheaper than the mammalian cell culture used to make larger antibodies.  

The nanobody technology platform has now been validated against more than 100 targets, including GPCRs, and in several animal models. "We are getting a lot of interest from some large pharmaceutical companies," says Allan. There are already three nanobody products in the clinic, which have made it in an unusually short time compared with conventional antibodies. One, licensed out to Wyeth, acts against tumour necrosis factor (an important molecule in inflammatory processes). Ablynx's own lead product, in Phase II, is a 'first-in-class' anti-thrombotic, a subcutaneous version of which is in Phase I testing. Meanwhile, another nanobody developed by Ablynx acts against receptor activator of nuclear factor kappa B ligand (RANKL) — the ligand of a receptor that is important in bone formation. The global biotech firm Amgen is also developing an antibody, denosumab, against RANKL for the treatment of bone loss and damage. Already in Phase III clinical testing, MorphoSys' Brkulj tips the agent as the next antibody blockbuster. The Ablynx product is a long way behind, but could it prove to be the first of a 'third wave' of therapeutic antibodies for an unmet medical need? 

Overall, the development of therapeutic antibodies remains an exciting area of research and it will be very interesting to see which firm will be the first to replicate or even better the success of the current blockbuster antibody title-holders. 

Articles

The identification of an increasing number of drug targets coupled with advances in manufacturing technology could lead to many more blockbuster therapeutic antibodies.

Therapeutic antibodies: the next generation

The UK Government has decided there might be money in biotechnology. As well as this, in a few weeks, the spotlight will be on what the world's most Northern scientific community can offer the biotech community with the 4th International BIOPROSP Conference on Marine Biotechnology (Norway). However, these developments have nothing to do with the human genome, monoclonal antibodies or biomarker-based diagnostics, as the biotech universe extends far beyond the biopharmaceutical advances — and setbacks — that hit the headlines.      

The biotechnology sector is occasionally described as a rainbow (with some colours missing) with each subsector having its own colour: red biotech is the medical sector, white (sometimes known as grey) the industrial sector, green is plant and environmental biotechnology, and blue is marinebased biotech. The term 'black biotechnology' is sometimes used to describe activities related to bioterrorism.  

All the sectors of biotechnology have something to offer the pharmaceutical industry. Big Pharma is using red biotech to fill its dwindling pipelines, which is all the more important as its 'patent cliff', a term analysts use to describe the unprecedented loss of patent protection drug makers face during the next few years, looms ever larger. According to a Datamonitor survey conducted in 2007, companies can expect to lose $140 billion (108 billion euro) in sales up to 2016 as their blockbusters face increasing generic, including biogeneric or biosimilar, competition. Therefore, pharmaceutical companies need biotech innovation from drugs hitting novel targets and pathways, such as RNA interference, therapeutic vaccines and even gene therapy. Hundreds of products similar to this are being developed in small biotech companies and academic departments worldwide. These SMEs need Big Pharma's cash and expertise to push their programmes towards the clinic, and so we can expect a steady stream of deals going forward into 2009. 

The application of white biotech to pharma may seem a bit less obvious. EuropaBio (the trade organization for the European biotechnology industry) defines white biotech as the application of biotechnology for the processing and production of enzymes, chemicals, materials and bioenergy. Much of the new interest in white biotech comes from the global drive towards a lower carbon, more 'knowledgebased' economy — both to try to alleviate global warming and to go easier on the earth's finite resources. Until now, the chemical, plastics, pharma and healthcare industries have been overwhelmingly fossil fuel based, both in their power sources and their production processes. This cannot continue. A big focus in white biotech is to find efficient ways to manufacture biofuels to replace fossil fuels. The other goal is to replace conventional chemical processes, including those using metal catalysts, with lower energy, cleaner, enzymebased processes.  

As mentioned previously, the UK government is taking an interest in white biotech. It has set up the Industrial Biotechnology Innovation and Growth Team, which is looking at the opportunities and challenges in this area. A policy document will be published later this year, setting out a strategy on white biotech.  

For the pharma industry, white biotech is well aligned with 'green chemistry' initiatives to make drug manufacturing processes leaner, cleaner, with less waste and toxics production. There is probably an enzyme for every chemical transformation you might want to do in assembling a drug molecule. The problem is obtaining that enzyme in the quantities needed for industrial processes. There are biotechnology companies, which include Biotec Pharmacon (Norway), Biocatalysts (UK) and Novozymes (Denmark), that specialize in doing just this — cloning a gene of interest, expressing the enzyme it codes for (and perhaps engineering it for better properties), scaling up production, and packaging and marketing it for the industry sector that can use it as a tool.  

The 'genetokilo' approach is the focus at the Centre of Excellence for Biocatalysis, Biotransformations, and Biocatalytic Manufacture (CoEBio3),

				  which is a consortium between the Universities of Manchester, Strathclyde, York and Heriot-Watt (UK). There is a wealth of research going on here into processes of interest to pharma, which is why AstraZeneca, Dr Reddy's, Merck and Pfizer are among the industrial affiliates of CoEBio3. Projects include methods for combinational biosynthesis of calciumdependent antibiotics (a new type of drug), with the identification of relevant enzymes on the biosynthetic pathway; the use of whole cell biocatalysts for the more efficient production of recombinant protein drugs; and biocatalysis in ionic liquids, which are of great interest as alternatives to toxic organic solvents in the pharma industry. 

Many years ago, I wrote a story about the production of blue dye indigo from bacteria. This was to replace the conventional chemical production of this important product. In 2008, this is still not a viable large scale industrial process — although it is getting there. This is the challenge with white biotech; companies don't want to change their processes unless they are sure the biological approach can be scaled up and will work. This is where the National Industrial Biotechnology Facility (NIBF), sited at the Centre for Process Innovation in Redcar (UK), comes in. Opened last year and working in close partnership with CoEBio3, the NIBF is a 'test bed' where companies — and they have pharma companies among their list of clients — can manufacture trial quantities of a material or compound using a white biotech method to see if they can make a commercial case for large scale production. 

One of NIBF's interests is in marine biotechnology, which brings us neatly into the blue biotech sector. Norway has a long tradition in fishing and exploiting the marine environment and, of course, it has plenty of oil. In recent years, attention has been shifting to bioprospecting — the search for compounds of value to the medical and food sectors in the deep waters of the country's fjords or in the more extreme environment of the Arctic. That is why bioprospecting, backed up with a wide range of genomics and molecular biology techniques, is becoming increasingly important for the world's most northerly university, the University of TromsØ (Norway). In February 2009, TromsØ will host a conference on bioprospecting where organizations such as the US National Cancer Institute will show their interest in this area in the hope that the sea might yield new cancer drugs.  

Again, pharma needs to fill its pipeline. There is some disillusion with the output from combinatorial chemistry, which can produce millions of potential drug leads in one go, but has failed to reduce attrition rates. Organisms, particularly bacteria and small invertebrates that are adapted to extreme environments such as the cold waters of the Arctic, may be a better bet when it comes to finding the next blockbuster or, to be more realistic, high value drug. Biotec Pharmacon is also located in TromsØ and has built a company specialized in the production of enzymes that can deal with extreme environments. Unlike conventional enzymes, which are sensitive to the conditions under which they are used, those that can survive unusual conditions have something extra to offer and molecular analysis can show which features of a molecule are adapted. These findings can then be used as a basis for molecular engineering to produce designer enzymes for a range of processes.  

There is no really obvious connection between the main area of green biotechnology, which is concerned with genetically modified crops, and pharmaceuticals. However, green biotech could use microbes and plants to clean up contaminated land and water. Such biological approaches could help industry deal with waste disposal. 

And finally, to black biotechnology. In the wake of 9/11, the US Department of Health and Human Resources handed out several contracts to companies to mass produce vaccines against tularaemia, plague and smallpox, which are all thought to be potential terrorist weapons. In April 2008, the Cambridge (UK) company Acambis won a £213 million (€244 million) contract from the US government to supply smallpox vaccine. The cash injection will help them progress work on a vaccine against the hospital bug 

. Perhaps it's regrettable that a terror threat represents a business opportunity, but many patients could benefit in the end. 

1. Centre of Excellence for Biocatalysis, Biotransformations and Biocatalytic Manufacture. 

www.fiercebiotech.com/story/patent-cliff-looms-for-big-pharma/2007-05-02

The biotechnology sector is occasionally described as a rainbow, with each sub sector having its own colour. But what do the different colours of biotechnology have to offer the pharmaceutical industry?

The four colours of biotechnologyy

The Biopartnering Europe (BPE) conference always marks the start of autumn for me. It's a place where small biotech companies make their pitches (so you get to hear about some great new science) while Big Pharma gives presentations on trends in the business and what they're looking for. BPE is also a kind of dating agency — an opportunity to network and set up a meeting with someone you might possibly do business with. 

Journalists are not part of what goes on behind closed doors at these events, but I'd love to know how many of the hundreds of pharma–biotech deals, big and small, that have been announced in 2008 began at a meeting such as BPE. These conferences are expensive and take people away from the bench and boardroom, but they could be a worthwhile investment for small biotechs looking for the lifeline that a deal with Big Pharma often represents. There are also advantages for Big Pharma as a biotech deal helps fill the pipeline and can extend the global coverage of both companies. 

There are people who analyse deals, and those whose business it is to spot trends and movements. I'm not one of them, but I love listening to these people talk about the biotech business; where it's going and what the most significant deals of the year are. Here are a few that caught my eye during 2008. 

By the middle of 2008, several one billion-dollar deals had already been made since the start of the year.  

In April, GlaxoSmithKline (GSK) went for the acquisition of Sirtris Pharmaceuticals (MA, USA), which is only 4 years old. Sirtris is working on an intriguing new class of enzymes called the sirtuins that appear to play a role in the ageing process. Sirtris are trying to mimic some of the health benefits of calorie restriction by activating the sirtuins with small molecule drugs. It has mainly focused on type 2 diabetes, although the approach is applicable to other areas. The deal gives GSK's drug discovery business several new compounds for metabolic disease, neurology, immunology and inflammation to work on. 

Also in April, Roche acquired the small biotech Piramed (UK). Formed in 2003, Piramed has developed new oncology drugs that are small molecules targeting two different forms of PI3-kinase, an enzyme central to a pathway known to be important in disease progression in cancer and also in the regulation of certain immune-based diseases. 

In June, Bristol-Myers Squibb (BMS) acquired Kosan Biosciences (CA, USA), adding to its pipeline cancer drugs that belong to two new classes known as epithiolones and heat shock protein (Hsp)90 inhibitors. The epithiolones are described as microtubule stabilizers, which have potential applications in various cancers as well as neurodegenerative disease. In the Hsp90 programme, BMS now has a Phase III compound. 

Vaccine companies are increasingly attractive targets for Big Pharma. In July, the leading French pharmaceutical company sanofi aventis bought Acambis (UK). The two had already been working together to develop vaccines for Japanese encephalitis, West Nile virus and dengue fever — all emerging diseases where no adequate protection is currently available. Acambis was a good candidate for takeover, as the company won a 10-year contract with the US government for supply of the smallpox vaccine and is also working on vaccines for 

The significant deals are not all about Big Pharma acquiring small biotechs. There is also consolidation going on between the biotechs themselves. In September, BTG (UK) acquired Protherics (UK), creating the UK's biggest biotech to date and one that is likely to enter the FTSE 250. Protherics has a potential blockbuster in Cytofab, a treatment for sepsis, although clinical trial data on this will not be available until 2010. 

Commenting on the year's activities, Bill Kridel, Managing Director and founder of Ferghana Partners (NY, USA), a leading life sciences investment group, says: "A major theme in 2008 has been franchise build-up. People are looking to do better in their existing franchises, filling in the gaps, whether they be geographical or in their pipeline. All of the large deals are of this nature." 

He cites the acquisition in late 2007 of Adnexus Therapeutics (MA, USA) by BMS, which gives the bigger company access to new biologics, applicable to a range of therapeutic areas. Adnexus has been developing a new class of drugs called Adnectins, of which the lead compound is Angiocept, an anti-angiogenesis agent currently in early phase clinical trials. The adnectins are based upon the widely distributed human protein fibronectin that exists outside cells and is involved in various targeted protein–protein interactions. The adnectins can be seen as an alternative approach to antibodies when it comes to targeted therapeutics.  

Kridel also mentions the deal between Novartis and MorphoSys (Germany), whose HuCAL technology is applied to the generation of human antibodies for research, diagnosticsand therapy. The 10-year deal, signed late last year, gives MorphSys the option of participating in selected Novartis discovery and development programmes, with Novartis funding these joint efforts. Morphosys has just exercised the first of these options for developing a therapeutic antibody against a promising target.  

Meanwhile, Takeda (Japan) signed a deal with the RNAi company Alnylam Pharmaceuticals (MA, USA) in May. RNAi is an emerging biotech approach involving the 'silencing' of genes involved in disease. Under the agreement, Takeda will have the first option to develop and sell Alnylam's RNAi therapeutics in Asia, marking the globalization of the new technology, while Alnylam will develop and sell in the US any RNAi therapeutics that Takeda discovers. Takeda has been exceptionally busy in 2008. They also did a deal with leading biotech Amgen which gives it rights to develop 13 of the molecules in Amgen's pipeline including motesanib, a promising anti-angiogenesis small molecule for cancer therapy.  

In May, Takeda also took over Millennium Pharmaceuticals (MA, USA), one of the world's leading biopharmaceutical companies, which was founded in 1993 and is famous for the discovery of VELCADE (bortezomib), the first effective treatment for multiple myeloma. 

Deals are not always about innovation as generics, including biogenerics, are now an important sector of the market. Israel-based Teva, the world's biggest generics company, consolidated its position in 2008 by purchasing Barr Pharmaceuticals (NJ, USA). Back in January, Teva also announced it would buy CoGeneSys (IN, USA), a biogenerics company.  

Looking back, Kridel says the level of activity in 2008 has been similar to that in 2007. For 2009, there will be "more of the same". In particular, we should look out for deals by Japanese and Indian companies, such as Dr Reddy's (India) and Wockhardt (founded in India, but now based in the UK). "This is the way of the future," Kridel predicts. 

At the time of writing this, Roche is attempting to buy an even bigger share in Genentech (CA, USA), the world's leading biotech company, but it's not yet clear how much it will have to pay. Much depends on the outcome of new clinical trials for Roche's Avastin, which may lead to the drug being more widely applicable in the treatment of colon cancer, a development that could add billions of dollars in sales. There is also further activity from Eli Lilly, which has bought ImClone (NY, USA). 

How the current global turmoil in the financial markets affects the progression of these and other ongoing deals remains to be seen, but it is likely that building and consolidation between pharma and biotech will continue at a brisk pace throughout 2009. Watch this space!  

1. Fierce Biotech, "2008's Billion-dollar deals (H1)" (July, 2008). 

Hundreds of pharma&#150;biotech deals have been announced during 2008 and many of these probably began at biopartnering conferences. Susan Aldridge examines some of this year's big buyouts and mergers.

The dating agency

There are many challenges upstream and downstream in manufacturing a biotech drug. Research on the upstream side in recent years has produced gene expression systems that have allowed for dramatic increases in yields of monoclonal antibodies, new vaccines and recombinant proteins. Downstream developments are catching up, with products such as the disposable Rhobust column from Upfront Chromatography (Denmark) and GE Healthcare's PreDictor, which is a 96-well plate system for rapid testing of purification conditions such as pH and ionic strength. However, purification is not the end of manufacture — it is just the beginning of the end. 

The main challenge of getting a biotech drug from this stage to the patient is the delicate and unpredictable nature of protein molecules. According to Geoff Helleswell of Micropharm (UK), the manufacture of their anti-sera and polyclonal antibody products takes a couple of days, but formulation, filling and signing off a batch can take 4–6 weeks. The cost of formulation and filling, as well as ensuring stability, is a big issue for the company as it produces antivenoms for countries, such as Nigeria, that cannot afford expensive freeze drying, which is a popular formulation technology for protein drugs.  

"There are several stages involved in the formulation and filling of a biotech drug", says Helleswell. Some of them are similar to those for small molecule drugs, but regulatory authorities often demand more data for a protein drug because of its inherent instability, and various tests must be done on the bulk product, with bioburden (microbial content), protein concentration, and virus removal or inactivation being the most important. 

At this stage, the question arises as to whether the product can be terminally sterilized. For a biologic, the answer is usually 'no' as protein molecules unravel when exposed to the heat of a sterilization operation. Therefore, filtration into a previously sterilized container will be necessary, with a recommended second filtration step immediately prior to filling. "Double filtration is very important for biologics," says Helleswell. 

At this stage, the product is being exposed to the air so consideration must be given to the environmental conditions for filling. "The environment in which the filling is done is critical," explains Helleswell. An EU Grade C environment is fine if terminal sterilization is intended, but the higher Grade A is necessary if the product has to be filled under aseptic conditions, meaning that manufacturers, including CMOs, must invest in the appropriate clean room technology if they are going to do their own formulation and filling. 

Most biopharmaceutical products are injected. This means preparing either a freeze-dried solid product that is reconstituted in the clinic or solutions that are contained in either capped vials or sealed ampoules. Prefilled syringes, for ease of patient use, are an increasingly attractive alternative in some applications. Additives, such as salts or preservatives, might be necessary to stabilize the protein and keep it stable for whatever storage times and conditions are appropriate to the final end use. A great deal of development work is needed to select the most appropriate additives to ensure they do not give any untoward clinical side-effects. 

The filled product must be inspected for complete closure and for visible and subvisible contamination. Meanwhile, sterility is checked with samples that are representative of the whole batch. Other tests, such as protein concentration, pH, osmolality and activity may also be required.  

Visual inspection, which can be performed by eye by a validated operator or with an automated visual inspection machine using lasers, is an important check. Regulatory authorities have requirements for clarity and place limits on even subvisible particles in a product. Proteins tend to precipitate, adsorb onto surfaces, or aggregate — all of which can cause visible or invisible deposits.  

The clinical significance of aggregation processes — particularly whether they can cause unwanted immune side-effects — is currently a subject of some debate. No protein product can be guaranteed to be 100% free of aggregation, but it should be understood and controlled as much as possible for a particular process and product. Aggregation often goes undetected and the underlying causes can be quite complex. pH and ionic strength of the formulation mix tend to be important factors and freeze drying often causes aggregation in some products when they are reconstituted. Aggregation can also occur during transport of a product by the use of pumps and even certain types of packaging. Additionally, shaking a protein solution is less likely to cause aggregation than stirring it.  

Visual inspections are important for assessing formulation stability as well as protein aggregation. There are a number of approaches for assessing the particle content of a protein solution. Light extinction methods are used to work out the actual size of the particle, but they are not applicable if the particle is translucent. Sometimes the sample must be diluted, which can cause interfering air bubbles, and may, in itself, cause aggregation. 

Experts in bioprocessing use a variety of methods to detect the degree of aggregation in a particular product, which goes from the simple monomer to a visible aggregate particle; size exclusion chromatography remains the standard, although dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) are often used. DLS is good for examining larger particles, while AUC is well suited to resolving a mixture of particles of different sizes and can give a handy chromatogram-like result. However, neither method is easy to use or validate for batch sign off purposes. As regulatory authorities expect manufacturers to use more than one approach to investigate products, a combination of these technologies is being increasingly used in formulation development. 

One new approach that can be used in this context is Micro Flow Imaging (MFI), developed by Brightwell Technologies (Canada), which combines the speed of a particle-counting method with the visual insight of a manual microscopic approach. In MFI, a volumetric section of the protein solution is investigated so particles are counted, sized and imaged. It allows all kinds of particles, protein aggregate or not, to be measured and assessed. Protein aggregates can be tricky to study because they are fragile, highly transparent with irregular size and shape, but MFI can cope with these issues. In an example presented at a recent bioprocessing conference (Vienna, Austria; April, 2008) by the company, MFI was shown to be highly efficient in checking the effectiveness of a filtration step in particle removal, and causing (or not causing) aggregation to occur. In other experiments, MFI could detect particles formed when a protein solution was forced through a syringe.  

Once the batch has been formulated, it is on to the final stages — and the paperwork. Helleswell describes the subsequent labelling and packaging steps as a "minefield". Labels must be authorized by regulatory authorities and special attention paid to their position, particularly if they are being applied manually as is often the case if costs are to be driven down. Then, the final product must be assembled, complete with the patient information leaflet. The packaging must ensure that the product stays in good condition during storage and transport, which is more challenging for a biotech product than a small molecule.  

Then there is the paperwork (increasingly being done in paperless format). A batch manufacturing record (BMR) covers all records during production, labelling and packaging and quality control for inspection by the Qualified Person (QP). The QP certification includes review of the BMR, production conditions, stability reports for the expiry date, validation, GMP standards required and authorization/licences available. Afterwards, the product is finally shipped and distributed, but to complete the life cycle there must be some arrangement for complaints, returns and disposal to be dealt with.  

Many of the steps described are common to conventional small molecule drugs and biologics, but there are differences because most biologics drugs are proteins, which vary from batch to batch in a way that a synthetic chemical does not (or should not). This means that formulation and the final product should be considered early in manufacturing so that the potential heterogeneity of the batch itself — and between batches — is better understood and controlled. Helleswell says: "In general, biological medicinal products require enhanced in-process controls to ensure batch-to-batch consistency."

There are many challenges upstream and downstream in manufacturing a biotech drug.

The beginning of the end

In the popular view of nanomedicine, miniature robots equipped with a set of tools will one day patrol the inside of the body, cleaning up atheroslerotic plaque, zapping cancer cells, and generally performing repair and maintenance. This highly futuristic scenario owes much to a vision presented by Eric Drexler, the pioneer of nanotechnology, in the 1980s. In reality, nanomedicine is making a slow, but steady, impact on the pharmaceutical industry. In 2006, Nature Materials estimated that there were approximately 130 nanodrugs or delivery systems in development across the globe,

 which, although is not many compared with the number of small molecules and biologics in the pipeline, is enough to create an intriguing new product category. 

Nanotechnology involves engineering functional systems at the molecular scale. While Drexler is credited with the major development of the field, the concept can be traced back to the physicist Richard Feynman who, in 1959, said: "There is plenty of room at the bottom" when referring to the possibilities in the nanoworld.

 The nanoscale is really the molecular scale, with small organic molecules lying at the lower limit of this range, viruses towards the upper end, and protein molecules and other biologics somewhere in the middle. Nanotechnology is well established today in the computing, energy and electronics sectors, but less so in pharma and biotech.

Nanomedicine is, put simply, the application of nanotechnology to medicine. In its first issue, the journal 

 adopted a more precise definition of nanomedicine as 'the use of materials, of which at least one of their dimensions that affects their function is in the scale range 1–100 nm, for a specific diagnostic or therapeutic purpose.'

 Then there is bionanotechnology — a broad and sometimes vague term that refers to the biomedical or biological application of nanotechnology. This can encompass the use of biomolecules themselves as nanodevices. Nanobiotechnology is another term in common use, which although is hard to define precisely, is generally understood as the use of nanotechnology in biotech applications.

For the pharma and biotech industries, the advantages of nanomedicine lie mainly in its ability to make drugs with better pharmacokinetics and bioavailability. "Materials can have very different properties at the nanoscale," explains Dr David Sarphie, CEO of Bio Nano Consulting, a joint venture between Imperial College and University College (all UK) that advises on the development and commercialization of new biomedical products in the nanotechnology area. For example, carbon nanotubes, which are an allotrope of carbon made of long cylindrical tubes, are exceptionally strong compared with more conventional forms of carbon.

The nanomedicine products already on the market fall into five categories: nanoparticles, nanocrystals, dendrimers, liposomes and micelles. The Fraunhofer Institute for Manufacturing Technology and Material Research Applications (Germany) and Bio-Gate, Nürnberg, a spinout of the University of Erlangen (Germany), have revived silver, a traditional antimicrobial, in the form of nanoparticles. Their technology can be used to coat medical instruments and devices, wound dressings, lab coats and bandages to protect patients against infection. With antibiotic resistance reaching epidemic proportions, new approaches to fighting infection will be welcomed. In a related development, smelly feet can be overcome by wearing socks impregnated with silver nanoparticles that kill the bacteria responsible for foot odour.

Elan (Ireland) has commercialized its NanoCrystal technology, which involves reducing the size of crystalline drug particles into the nanoscale to increase the drug surface area and aid the dissolution of poorly water-soluble compounds, thereby improving bioavailability. The first nanocrystal product is a new version of the immunosuppressant sirolimus. As it is a solid dose formulation, it is more easily stored and administered than the previous oral solution that needed to be refrigerated. As patients are usually on sirolimus for life, the advantages are clear. Elan has also offered to help other companies put their products into nanocrystal form.

Another emerging class of nanodrug is the dendrimer, a precisely defined synthetic complex molecule. A dendrimer is synthesized around a core molecule with branching functional groups, to which other branching molecules can be added in layers to create a molecule with many functional groups on its surface. The nature of these groups can be tightly controlled and the advantage of a dendrimer is that it can activate many receptors simultaneously, whereas a small molecule will only interact with one receptor. Starpharma (Australia) is developing dendrimers based on the amino acid lysine (with its two branching amino groups) for a variety of applications including HIV, genital herpes, drug delivery and small interfering RNA delivery, and already has a vaginal microbiocide dendrimer product on the market for HIV, bacterial vaginosis, HPV and contraception.

Liposomal and micellar formulations of drugs bring them into the nanoscale, and have been used in a number of controlled-release systems including: Doxil (Alza, USA), a liposomal formulation of the anticancer drug doxorubicin; Medicelle (NanoCarrier, Japan), a micelle-based technology for anticancer drugs; and Basulin (Flamel Technologies, France), a long-acting insulin. Meanwhile, conjugation of drugs with polyethylene glycol (PEG) is another popular nanotech application that improves the half-life of drugs, such erthyropoetin for anaemia, and interferon-alpha for hepatitis C, meaning that the patient has to take fewer doses, which can improve the quality of life for the patient in terms of fewer injections.

Manufacturing nanomedicines is conducted either by breaking down a material (nanocrystals) or building one up (dendrimers); for example, Elan makes its nanocrystals by milling the drug substance using a wet-milling technique. The particles are prevented from sticking together by surface absorption of stabilizers, resulting in an aqueous dispersion that behaves like a solution and can be further processed into finished dosage forms. Purification is not as straightforward as for a small molecule and there will always be a size distribution in a PEGylated product as polymer molecules vary in length.

Whether improved purification methods will provide clinical benefits remains to be seen. "Pharma has not jumped headfirst into nanomedicine," says Sarphie. There are potential risks with particles at the nanoscale as it is still relatively unknown territory. A recent paper in 

 by Ken Donaldson and his team at Edinburgh's Safety of Nanomaterials Interdisciplinary Research Centre (UK) highlighted the potential hazards of the carbon nanotube.

  Donaldson points out that there are many resemblances between carbon nanotubes and asbestos fibres: they are both very thin (10–200 nm), are either long and straight or may tangle, and are very tough, durable and not readily soluble in tissue. The team examined cells lining the pleural cavity that are the target of mesothelioma in an animal model, a cancer caused by asbestos fibres, and found that long carbon nanotubes caused damage, inflammation and scarring of the mesothelium, similar to that found with asbestos. However, the short carbon nanotubes caused no damage. The work raises several queries regarding the safety of carbon nanotubes, but the question is would medicines based on carbon nanotubes have the same adverse impact on the mesothelium as asbestos? It has already been proposed that the nanotubes would be good for delivering short interfering RNA into cells to regulate gene activity, but the answers regarding safety are needed fast as the last thing the emerging field of nanomedicine needs is a catastrophe.

Meanwhile, another report suggests that silver socks may have an adverse effect on the environment.

  In experiments where socks impregnated with silver nanoparticles were washed, the particles leached into wastewater. Entering the water supply in this way could upset microbial ecosystems by killing off certain species of 'friendly' bacteria.

Despite the concerns, there is still plenty of room for innovation in nanomedicine beyond drug delivery applications and for investment, and some of the benefits may come from new drug discovery and development tools rather than nanodrugs themselves.

1. Editorial, Nanomedicine: A matter of rhetoric?, 

2. R.P. Feynman, There is plenty of room at the bottom (1960). 

4. K. Donaldson, Commentary on Nature Nanotechnology Paper (2008). 

www.safenano.org/Uploads/Donaldson_NatureNanoCommentary.pdf

5. P. McKenna, Smelly sock treatment leaks silver nanoparticles (2008). http://

technology.newscientist.com/channel/tech/nanotechnology/dn13602-smelly-sock-treatment-leaks-silver-nanoparticles.html

In the popular view of nanomedicine, miniature robots equipped with a set of tools will one day patrol the inside of the body, cleaning up atheroslerotic plaque, zapping cancer cells, and generally performing repair and maintenance.

It's a small world

  Researchers at Children's Hospital Boston (MA, USA) recently announced another group of stem cells that can produce cardiomyocytes (heart muscle cells). It seems hardly a month goes by without some kind of discovery in the fast-moving world of stem cells. The latest announcement opens up the prospect of treating heart failure with cell therapy. This is important as heart failure, marked by the loss of cardiomyocytes, is on the increase and the only successful treatment is a heart transplant.   

Stem cell therapy for heart failure is many years away, but cardiomyocytes, as well as other stem-cell-derived cells, could help patients by providing pharma companies with a powerful new tool for producing safer and more effective small molecule treatments.  

In recent years, companies have come to increasingly rely on cell-based assays to provide them with physiologically relevant information at an earlier stage in the value chain in the hope of decreasing the attrition rate of compounds as they progress from bench to clinic. They are now looking at developments in imaging, molecular biology and stem cell technologies to provide even better cell-based models. For suppliers, meeting pharma's demand for large amounts of cells for assays is an excellent learning experience for the time when they will have to manufacture them for the clinic. 

A few years ago, pharma companies began to move towards cell-based screening, starting off with standard cell lines, such as chinese hamster ovary cells or HeLa, a cancer cell line. Today, cellular assays probably account for more than half of all high-throughput screens, and these methods are also being used in secondary screening and lead validation work. "We are seeing requirements for better assays in more relevant cell models," says Ger Brophy, General Manager of Advanced Systems at GE Healthcare in Uppsala (Sweden). GE has devised a service that not only provides cells and related products, but also works with customers on assay development and related issues. There has been an increase in requirements for frozen cells, which are according to Brophy, beginning to be seen more like a reagent than a specialist research product by pharma and biotech customers. 

Not only do customers want cells to be similar to chemicals in their consistency and ease of use, they also want them to work harder. The high-throughput concept is well-established and the buzzword now is 'high content', with the maximum amount of information being extracted from each experiment in a cell-based assay — a development that relies on increasingly sophisticated imaging and software systems. 

"A cell is basically a bag of biochemical pathways," says Tim Allsopp, Chief Scientific Officer of Stem Cell Sciences (SCS) in Cambridge (UK), which manufactures and delivers physiologically relevant cells derived from stem cells to the pharma industry. "Cells can be genetically modified to tell researchers what is happening to them. For instance, reporter molecules can be engineered into a gene pathway so that they light up if that pathway is activated. There has also been a move to high-content analysis of cells containing a plurality of reporters, labelling a number of distinct biochemical pathways so that researchers can glean a range of biological information." 

SCS works with tissue-derived stem cells, from which they obtain neural, muscle, fat and bone cells, and both mouse and human embryonic stem cells (ESCs). "Companies are used to having a variety of reagents and now they want a range of cell types," says Allsopp. "Many researchers use mouse cells, but, ultimately, drugs are designed for humans and so should be tested on human cells."  

Put simply, stem cells are important for screening because they can be indefinitely propagated. If this process can be properly controlled, then mass production should be possible, along with differentiation into primary cells. Previously, as Allsopp points out, cells used in screening were made from abnormal tissues, including cancer cells, with abnormal biochemical pathways — so how relevant were the data from them? 

Fergus McKenzie, Programme Manager of ITI Life Sciences (UK), which is partnered with Cellartis (Sweden), adds: "It is always better to derive an assay in a physiological cell type." Therefore, human ESC-derived hepatocytes (liver cells) are better than those derived from cadavers and cardiomyocytes are now being used as well. "My belief is that you will get better toxicology data from these cells," says McKenzie, explaining that it is not ESCs themselves that companies want, but the cells they can turn into by differentiation. Currently, the progenitor cells of hepatocytes and cardiomyocytes are the most advanced, with pancreatic cells following. There is also interest in getting vascular cells to work in combination with cardiomyocytes in screening models.  

The challenges on the suppliers are to scale-up and provide a consistent supply of cells, including stem cells, to make them more like chemical reagents. "We are focused on consistency of supply," says Allsopp. "The cells we supply should have reproducible properties as 'drift' in the cell properties during a screening campaign would make data interpretation problematic. Ramping up supply to a customer's needs can be challenging, even for a commercial organization such as ours with automation, but it is a trend for the future so we must optimize our processes." 

Brophy adds that at GE Healthcare's facility near Cardiff (UK), a number of rooms of the appropriate size have been established for large-scale production. Cytodex, a bead-based product from GE Healthcare Life Sciences, is proving important as it provides a hugely increased surface area within a bioreactor to which the cells can adhere. The company has also responded to the growing demand for cells that have been transiently transfected rather than exhibiting stable expression. Transient transfection is difficult to scale-up using traditional methods, but GE Healthcare is developing methods for mass transfections in bioreactors rather than in batches. This process should be more efficient and improve consistency.  

The other consideration in the large-scale production of cells is the format in which they are supplied and distributed to customers. Ease of handling is important and so is miniaturization where a lot of information can be extracted from a small amount of material, which means cells plated onto a microwell plate are popular. "Customers want to treat cells as a consumable that you use and then dispose of," says Allsopp.  

McKenzie adds that stem cells for therapy, as are being used in clinical trials by ES Cell International of Singapore and Geron (CA, USA), must be at GMP standard, but this is not needed when the cells are used as a drug discovery and development tool. "Stem cells are still expensive and a technically advanced product," he says. "There is not a commodity market in stem cells at this time and it will be a few years before their use in drug discovery is widespread." 

There is some way to go before all current opportunities in cell-based screening are exploited as it is still too soon to show data proving that such screens do, indeed, lead to reduced attrition rates. "There is resistance to change in Big Pharma and smaller biotechs don't have enough money to invest," McKenzie says. However, Pfizer (NY, USA) has apparently been working with mouse ESCs for some time and turning them into a range of cell types for screening. Other companies are likely working with ESCs 'on the quiet'.  

In the future, besides a move to stem cells, McKenzie predicts: "There will also be a move to two-dimensional cellular models with monolayers and more complex structures on scaffolds and maybe even small organs." Brophy adds: "There will be advanced cell models that can bring together validation and toxicity. The complexity of the body will be reflected in the type of cellular assay."

Researchers at Children's Hospital Boston (MA, USA) recently announced another group of stem cells that can produce cardiomyocytes (heart muscle cells). It seems hardly a month goes by without some kind of discovery in the fast-moving world of stem cells.