Susan Aldridge

While it's unlikely that Al Gore's Nobel Peace Prize will gain him the US Presidency, the award has put global warming firmly in the spotlight (as if it wasn't there already). Reducing its carbon footprint is among the many goals Big Pharma has for the coming year — and beyond. Many energy consumption issues are common to all industries and individuals, such as putting curbs on business travel, bringing renewable energy sources on board and switching off lights in unoccupied rooms, but there are other issues that are specific to our industry. For example, asthma inhalers emit greenhouse gases and many pharmaceutical syntheses use toxic solvents. 

Similar to all businesses, pharmaceutical companies face challenges in reducing their carbon footprint, such as changing company culture and making a business case for investing in new technology that could take time to show a return. But the potential benefits are undeniable; by reducing their carbon footprint, companies help the planet and improve their image (always a bonus for pharma), and reduce costs, thereby pleasing shareholders. 

I took a quick peek at the approaches being used by a couple of the big companies, to see if they really are leading by example. The plans appear to be comprehensive and ambitious. There are car sharing schemes, wind and solar power, green facility design, and plenty of green chemistry — to name but a few of the ideas underway. Green chemistry, in particular, is being used to unite the companies in their search for cleaner, leaner pharmaceutical production. 

The American Chemical Society (ACS) Green Chemistry Institute created the Pharmaceutical Roundtable in 2005 to "bring key medicines to the patient with minimum impact on the environment". The Roundtable is open to all pharmaceutical research, development and manufacturing companies, and currently comprises AstraZeneca, Eli Lilly & Company, GlaxoSmithKline (GSK), Johnson & Johnson, Merck & Co., Pfizer and Schering-Plough Corporation — all of which have their own green chemistry programmes. At their last meeting, they agreed on a number of areas where better chemistries need to be developed. A major objective was to reduce the number of processes that involve the formation of amide bonds and the reduction of amides, both of which are prominent in pharmaceutical chemistry. Forming an amide is a reaction with 'poor atom economy' — that is, to put simply, it generates too much waste. Reduction of amides generally involves hydrides that give rise to waste byproducts that need special treatment. They also want to reduce the use of chlorinated solvents and search for solventless methods of cleaning equipment. 

Being the first pharma company to appoint a full-time green chemistry leader, Pfizer takes its obligations very seriously. In 2003, the company won a Crystal Faraday award for work on 'greening' the synthesis of sildenafil (Viagra), followed by an AstraZeneca award for Excellence in Green Chemistry and Engineering for pregabalin (Lyrica). Pregabalin, a treatment for nerve pain, is a significant new product for the company and will account for 15–20% of API production by 2010. The award-winning research involves implementation of a biotransformation process for pregabalin synthesis, where all four steps take place in water. ''This will lead to a significant reduction in our use of carbon-based solvents,'' says Peter Dunn, Pfizer's green chemistry leader. "It is also very unusual to have as many as four steps performed in water." The process will lead to a decrease in solvent wastes requiring incineration. Calculations also suggest that the new way of synthesizing pregabalin cutsin energy and material inputs by a fifth compared with the first generation synthesis at the 1000 kg scale. 

The achievement underlines the company's commitment to biotransformations, with dedicated teams of microbiologists in the US and UK looking for new ways of using enzymes in manufacturing. Dunn adds that all Pfizer R&D sites have a green chemistry team comprising medicinal chemists, process chemists and safety specialists, and there are also five PhD students working solely on green chemistry themes for the company. They have annual conferences at each research site and hand out 13 dedicated awards, which are given to charity or to green chemistry researchers in academia. Pfizer is also involved in running workshops globally to interest undergraduates: "We are trying to promote green chemistry to a new generation of chemists," says Dunn.  

Similar to other Big Pharma companies, Pfizer takes a very broad view on the carbon footprint issue and has been adopting an 'aggressive' and global strategy to reduce emissions since 2002. "We look at anything and everything that generates greenhouse gases in our operations," explains Charles Calitri, senior director of engineering operations and technical services, Pfizer Global Engineering. "Our focus has been on direct and indirect emissions from our site operations and we are now beginning to assess impact outside our operations such as those emissions resulting from business travel, our fleet and contributors along the supply chain. Although it has sometimes been hard to quantify the impact in terms of carbon reduction, we are now getting closer to the full picture." The company is committed to reducing greenhouse gas (GHG) emissions and is setting a second generation goal after achieving its original GHG reduction goal in 2007. In addition, Pfizer intends to meet 35% of its global electricity needs by 2010 through 'clean' energy sources. Teams at Pfizer use a number of innovative electronic and knowledge transfer tools, such as a web portal listing more than 3000 different projects related to energy issues to quantify energy savings and knowledge sharing. Pfizer is also committed to develop environmentally responsible facilities through its green building programme, conserving natural resources and reducing energy consumption.  

The company has undergone some dramatic changes in its business model in recent years, but its environmental programme has survived the pressure. "People are becoming more accepting of the need for real change when it comes to energy consumption," says Calitri. What has probably made the difference is the company's appointment of 'energy champions', who are dedicated to accomplishing the 'green' targets at both regional and site level, as well as the public commitment of senior figures to the carbon reduction strategy. Calitri adds that it is vital to maintain an external focus to their efforts through alliances with academia, industry and organizations such as the International Society for Pharmaceutical Engineering, which is forming a new group for carbon issues. "We are trying to tap as many sources as we can," he says.  

Meanwhile, James Hagan, vice president corporate environment health and safety at GSK says they go right back down the supply chain when it comes to carbon footprint. "We look at energy conservation as an organization — from R&D, manufacture and distribution to transport and the movement of employees." One key issue is emissions from inhalers. GSK spent one billion dollars looking at alternatives to chlorofluorocarbons and came up with 1,1,2,2 tetrafluoroethane (HFC134a). 

It still has some global warming potential, so the company is looking for alternatives to this as well. GSK's new asthma drug Seretide is available in a dry powder formulation, but this is not suitable for all patients, so it is not possible to avoid propellants completely. Indeed, a substantial proportion of GSK's carbon footprint does still come from inhaler use. 

But the good news is that the company's carbon footprint has gone down year on year from the mid 90s. In 2000, the company set a target of getting carbon emissions down by 8% per unit of sales — and managed to exceed this with a reduction of 13%. "In 2007, the International Panel on Climate Change stated that they are 90% certain climate change will happen. We took this as a clarion call and stepped up our programme with aggressive energy and global warming targets," Hagan says. These comprise a 20% reduction in carbon emissions per unit sale by 2010 and 45% reduction by 2015. To do this, GSK has created a unique $75 million Special Fund to support initiatives such as the introduction of combined heat and power as an energy source. The company wants to be transparent about its global warming strategy and Hagan has invited me to return in a year's time to see where they are in achieving their goals (and I promise to do so). "We don't want to cover up if we don't make it," he says. 

He adds that the GSK plant at Barnard Castle (UK) has been very proactive and has put up two large wind turbines that generate 10% of energy needs. The site has also looked rigorously at the small things — turning off lights when people are not in a room, using low-energy light bulbs and equipment such as variable speed motors, which use less energy when rotating at lower speeds and can be adjusted to the nature of the task. In Singapore, the GSK plant gets 5–10% of its energy from solar panels, making it the country's most significant user of this form of renewable energy. Meanwhile, R&D is looking for a decrease of 50% in waste from all its products. "This is a major initiative. There are some real heroes in R&D," says Hagan. 

What do they hope to achieve by these efforts? "In 1995, the goal of carbon reduction would have been to save money, but concern regarding the company's obligations as a corporate citizen has become increasingly important. In 2007, money saving and the ethical aspect of carbon reduction are equally important to us. We have external stakeholders who have asked us to be more aggressive in our approach," says Hagan. 

And surely it is appropriate that human healthcare businesses are committed to improving the health of the environment as well?  

Articles

While it's unlikely that Al Gore's Nobel Peace Prize will gain him the US Presidency, the award has put global warming firmly in the spotlight (as if it wasn't there already). Reducing its carbon footprint is among the many goals Big Pharma has for the coming year - and beyond. Many energy consumption issues are common to all industries and individuals, such as putting curbs on business travel, bringing renewable energy sources on board and switching off lights in unoccupied rooms, but there are other issues that are specific to our industry. For example, asthma inhalers emit greenhouse gases and many pharmaceutical syntheses use toxic solvents.

Pharma going for a greener New Year

In recent years, Big Pharma companies have shown increasing interest in setting up manufacturing in India and the Far East. The advantages of this outsourcing trend are obvious — decreased costs, coupled with ever increasing standards of operation. However, as the technological and quality of life aspirations of these new locations increase, the attractive cost advantages could dwindle and cause pharma companies to look elsewhere for manufacturing locations. Perhaps, they will consider Africa. 

This is an interesting prospect. Public perception has long been that drug companies have neglected Africa — first by failing to invest much in malaria and other tropical diseases and, more recently, HIV/AIDS. They are seen as being preoccupied with the maladies of the affluent West: cancer, heart disease, diabetes and obesity. Even when they invented antiretroviral drugs (ARVs), making AIDS a chronic disease rather than a death sentence, the prices put them far beyond the reach of those who needed them most. HIV/AIDS claimed approximately two million lives in Africa in 2006 —several years after ARVs were introduced onto the market. 

However, AIDS has finally concentrated the minds of Big Pharma upon Africa, and initiatives happening there today might just form the basis for building manufacturing, and more, on the continent in the future. Companies can always adopt the humanitarian principle of distributing ARVs and other needed drugs free of charge, or at preferential prices, from existing manufacturing locations. Some good has already come from such schemes; Merck's free distribution of ivermectin has saved more than half a million people in West Africa from river blindness, but this approach now seems paternalistic when the pharma business, and so much else, has gone global. 

Pharmaceutical pricing, intellectual property (IP) and licensing are difficult issues for an industry suffering a productivity crisis in relation to its R&D investment, and these all impinge on improving healthcare in Africa. Despite this, there are now several ARV manufacturing partnerships underway in African countries between Big Pharma and local companies, according to Guy Willitts of the Geneva-based International Federation of Pharmaceutical Manufacturing Associations (IFPMA). These are described in the IFPMA's report '

Partnerships to Build Healthier Societies in the Developing World'

. Such operations could mark the beginnings of a pharma industry in Africa. 

Some companies have issued voluntary licences to allow local manufacturers to make cheap generic versions of their ARVs. This type of arrangement works best in locations where there is a severe HIV/AIDS epidemic, but possesses an adequate healthcare infrastructure, a relatively stable local economy and appropriate manufacturing expertise. The parent companies want to be sure that the local operation can ensure a long-term supply (ARV treatment is usually for life) of high-quality drugs that will go to those who need them. Safeguards against both counterfeiting and diversion of product to wealthier markets need to be addressed in this situation as well. 

According to the IFPMA report, Bristol-Myers Squibb now has a tech transfer agreement with generic company Aspen PharmaCare of South Africa on its latest ARV, atazanavir, which has involved IP and technical know-how related to manufacture, testing, packaging, storage and handling of the API and finished dosage form. Aspen is now working on regulatory submissions for sub-Saharan Africa. 

Meanwhile, GlaxoSmithKline (GSK), the world's largest supplier of HIV/AIDS drugs, granted its first voluntary licence in 2001 and now has eight such agreements for its ARVs in Africa, which includes one with a company in South Africa. Last year, GSK significantly increased manufacturing capacity in Africa to supply larger amounts of ARVs at lower prices and it is currently the only Big Pharma company that manufactures products in East Africa. Its regional manager John Masunga is also chairman of the Kenyan Association of the Pharmaceutical Industry and there are now similar organizations in Morocco and South Africa, suggesting that the foundations of a new industry are being built — at least in some parts of Africa. 

In 2005, Merck granted a nonexclusive, royalty-free patent licence for manufacture and supply of a generic version of its ARV, efavirenz, to Aspen PharmaCare, while last year Roche committed to a new 'AIDS Technology Transfer Initiative' to help manufacturers in the Least Developed Countries (as defined by the United Nations) to produce their HIV protease inhibitor saquinivir. In September 2006, they announced tech transfer agreements with three African companies.

Meanwhile, the Association of British Pharmaceutical Industries recently published a report 'Global Health and the Pharmaceutical Industry' that suggests work upstream and downstream of pharmaceutical manufacturing is also occurring in Africa. One example is Pfizer's project with the World Health Organization's Special Research Programme for Research and Training in Tropical Diseases (WHO-TDR), which provides WHO-TDR access to a section of Pfizer's compound library for screening against a range of tropical diseases. African scientists are also working alongside people at Pfizer to analyse screening results to select compounds worthy of further development. These skills can be transferred back to their home institutions to build up their tropical disease research capacity. Pfizer is also partnered with Makerere University (Kampala, Uganda) international infectious disease experts, the government of Uganda and a number of nongovernmental organizations in the Infectious Diseases Institute (IDI). The aim of this initiative is to strengthen local capacity in HIV/AIDS care and train doctors from several African countries in the often complex business of administering ARVs. IDI houses a state-of-the-art diagnostic lab that helps monitor HIV therapy and diagnose the opportunistic infections that often accompany HIV, as well as tropical and sexually-transmitted infections. Meanwhile, AstraZeneca is working for better diagnosis and management of breast cancer among women in Ethiopia, Abbott is funding paediatric AIDS clinics in Africa and elsewhere, while Novartis, through its Foundation for Sustainable Development, is working with the WHO to eliminate leprosy through education, diagnosis and distribution of multidrug therapy.  

Of course, the pharmaceutical industry could — and should — do more in Africa. The healthcare needs of the population must come first and fulfiling this goal could also make Africa a place where drug discovery, development and manufacture are conducted. This would help the industry with its global aspirations and could even improve its public image.  

In recent years, Big Pharma companies have shown increasing interest in setting up manufacturing in India and the Far East. The advantages of this outsourcing trend are obvious - decreased costs, coupled with ever increasing standards of operation. However, as the technological and quality of life aspirations of these new locations increase, the attractive cost advantages could dwindle and cause pharma companies to look elsewhere for manufacturing locations. Perhaps, they will consider Africa.

Is pharma moving to Africa?

If pandemic flu strikes this winter — and many health experts believe that such a catastrophe is only a matter of time — then vaccine companies will be under huge pressure to deliver supplies safely and on time. The news on the technology front is that US-based companies Protein Sciences and Novavax may be able to deliver the goods in less than 12 weeks after the start of a pandemic, using a new approach to vaccine manufacture involving recombinant DNA. This is interesting because it's only quite recently that players such as Novartis and GlaxoSmithKline announced big investments in animal cell technology for vaccine manufacture. It's generally acknowledged that the animal cell approach is safer, faster and more reliable than the traditional method of producing vaccines in chicken eggs (to say nothing of the logistics of handling thousands of eggs and managing the associated flocks of chickens). But could animal cell technology now be superseded by the recombinant DNA method in the race to get safe and cost-effective vaccines to the public? 

Choosing the right technology for manufacture is only one of the demands on vaccine companies. While public attention tends to focus upon whether we have enough shots for a pandemic of the deadly strain of bird flu (H5N1) or whether we are prepared to deal with a bioterror outbreak of smallpox, there are many other opportunities in the vaccine sector. For instance, we should not forget the World Health Organization's bid to eradicate polio from the planet by universal immunization (much as it eliminated smallpox) or the constant fight against childhood diseases such as diphtheria, measles, rubella and whooping cough (pertussis). Vaccines are now being formulated against cancer such as Merck's cervical cancer vaccine Gardasil. Meanwhile, researchers are looking for effective vaccines against HIV/AIDS and hepatitis C, and believe they can even immunize against nicotine and cocaine addiction in the near future. Different vaccines, different markets and many challenges for manufacturers.  

While innovation is the watchword in small molecules and most biologics, the world of vaccines tends towards conservatism. That is because the drivers of the market are somewhat different: vaccines are used on very large numbers of healthy people, many of them children. If a product is known to be safe — however old — then there will be little incentive for change. Therefore, the vaccines against tetanus and pertussis have remained more or less the same for many years. Nor is there really any room for generics or biosimilars. Yet, there are some sophisticated vaccines around too. Dr Crawford Brown, CEO of Eden Biodesign, which runs the National Biomanufacturing Centre (NBC) at Speke, near Liverpool (UK), points to Merck's Recombinax vaccine against hepatitis B as an important product. "The advent of the hepatitis B vaccine has to be seen as a seminal change for the industry. It is a well-defined product, which makes money, has a healthcare benefit and is safe," he says. "Merck and Smith Kline got a lot of momentum from showing that a recombinant vaccine could work and making it in yeast gave an efficiency of cost." 

Brown, who has long experience in vaccine manufacture and is now supervising some vaccine projects at NBC, says that more recent inspiration can be found in Wyeth's Prevanor, which protects against seven strains of Pneumococcus infection. "This has probably been the most successful launch of any biotech product," he says. "It is a very complex product because it requires the isolation and blending of seven different strains. But it is a $1 billion product — one of biotech's few blockbusters."  

Vaccine manufacture is broadly similar, in principle, to other biotech manufacturing operations, but with some important differences. For instance, it is generally done on a smaller scale because only microgram quantities are needed, and, therefore, disposables, rather than stainless steel, tend to be used throughout the manufacturing process. Process validation, product characterization and purity are particularly important in vaccine manufacture: "Purity is a particular challenge for vaccines, compared with small molecules," says Brown. "Some vaccine impurities have an immunomodulatory effect and act more like an adjuvant to the vaccine." In other words, vaccine impurities are more akin to an API than a mere nuisance that must be eliminated. Therefore, manufacturers have to think how to scale-up their production without losing the potency of the vaccine plus impurity mixture.  

Vaccines also differ from conventional drugs in that they are generally stockpiled and are not used continuously. People do not worry about whether there is enough aspirin or atenolol to meet demand — it is made and consumed all the time. But they are concerned about whether there is enough smallpox or flu vaccine ready to meet a sudden surge in demand. Therefore, vaccine manufacture is flexible, rather than continuous. "You may need to produce a batch of vaccine that is then to be stored for several years," explains Brown. "The nature of production is discontinuous compared with other products." Some of these products go back a long way and there may be items of knowledge involved in their production that are hard to document in GMP and the chance of the people in the know being around when an old vaccine is next needed is small.  

Meanwhile, cell banks required for vaccine manufacture may become depleted with time and a new working cell bank may be needed. "A working cell bank is rather like the Crown Jewels," Brown observes. "This is very different to small molecule manufacture where you are only concerned about raw materials — there is no equivalent risk. Expecting the cells to grow and perform as they did 10 years ago is a big ask." 

But growing expertise in cell culture technology and bioprocessing is helping manufacturers to overcome these problems. Therefore, it should be possible to put many new vaccines into robust large-scale production during the next few years — not only to protect against infection, but also against cancer and other conditions as an alternative to conventional pharmaceutical drugs. 

Vaccines are needed against old and new infectious disease threats - polio and other childhood illnesses, bioterrorism and pandemic flu. They are also emerging for cancer immunotherapy and for treating addiction. While vaccines are among some of the most successful biotech products, their large-scale manufacture involves some special demands, such as maintaining a good working cell bank and gearing up for production on an 'as needed' basis.

Pandemics to polio — challenges in vaccine manufacture

A pharmaceutical plant may seem a long way from the clinic, but everything that happens there is — or should be — guided by considerations of patient safety. In March 2006, six healthy volunteers dosed with the monoclonal antibody TGN1412 at a clinical trials unit at Northwick Park Hospital (London, UK) suffered sudden and extreme adverse reactions — an unprecedented event in a Phase I trial. The resulting headlines did the biotech industry no favours at all, and TeGenero, the German company that manufactured the drug, folded last July.  

MHRA accordingly set up an enquiry, which showed that no manufacturing issues were involved in the TGN1412 incident. So this differed from the viral contamination of blood products that infected thousands with HIV or hepatitis C. This scandal led, eventually, to requirements for biologics manufacturers to insert purification steps that assure viral decontamination into their processes; it also started a move away from the use of animal-derived components in manufacturing (a trend accelerated by the BSE crisis). Had some overlooked aspect of manufacture or purification put patients at risk again? No, as it turned out. Instead, the TeGenero problem derived from the novel mode of action of TGN1412, which was designed to 'superactivate' T cells in a way that was hoped would prove useful as a therapy for rheumatoid arthritis and leukaemia. The result in this case, however, was a so-called 'cytokine storm'. I'm no immunology expert, but I'm told this phenomenon is as unpleasant and dangerous as it sounds, involving an uncontrolled release of inflammatory molecules (cytokines) into the bloodstream, which leads to pain, vomiting, burning fever, massive swelling and multiple organ failure.  

MHRA's investigation has led to 22 recommendations on what more should be done in the transition from preclinical to clinical for certain biologic drugs. These have now been largely translated into the EMEA Committee on Medicinal Products for Human Use 'Guidelines on requirements for first-in-man clinical trials for potential high-risk medicinal products'. These are intended to provide a common approach across EU Member States for the design and conduct of such trials, although they are not binding. High risk refers to molecules with a novel mode of action, such as TGN1412, and also to those involving novel targets, as well as those for which there is (as yet) no relevant animal model for preclinical work.  

It is likely that the biotech industry will produce many more potential drugs in the first two categories; many companies are developing therapies that work on T cells and the Human Genome Project has generated thousands of new targets, many of which are regarded as 'novel'. We have also moved away from the traditional monoclonal antibody towards more novel, engineered molecules. The guidelines mention, for instance, bispecific antibodies and novel fusion proteins as being categories which may be regarded as 'high risk'. In short, the number of 'high risk' molecules in the biotech industry pipeline is sure to increase, with implications for both clinical trials and manufacturing.  

Many of the recommendations in the guidelines concern preclinical work and the design of clinical trials with high risk biologics. But manufacturers could also be affected if they are producing these molecules — especially in two of these areas we have touched on here before, namely, characterization and comparability. A high degree of quality characterization is now being called for at an early stage of development. In particular, characterization of product-related variants, including heterogeneity and degradation products, should be performed if these might have an impact on the molecule's pharmacological profile. The guidelines also ask for 'special consideration for the suitability and qualification of methods to sufficiently characterize the active substance and drug product'. More work for the analytical team, in other words. 

On comparability, the EMEA recognises how modifications to manufacturing processes can result in subtle changes to molecular structure that are not readily detectable by characterization studies — but may still affect biological properties and, therefore, have clinical consequences. This may lead to a requirement for more preclinical testing to try to prove comparability between batches made by different processes. Yet it is not clear how valid preclinical work actually is for some of these novel biologics; tests in monkeys gave no hint of the problems to come with TGN1412.  

The guidelines were published in March after which there has been a two-month consultation period, followed by a stakeholder workshop in June. At the time of writing, it is not clear what kind of agreement will be reached. If they remain as guidelines, then EU Member States will interpret them as they see fit. At a recent conference, Professor Gordon Duff of the University of Sheffield, who chaired the MHRA investigation into the TGN1412, said they were determined not to inhibit biotech innovation. I wonder.  

In February, MHRA announced new rules for clinical trial applications involving novel biologics; these must now first be checked by a specialist advisory group. This is likely to slow down entry of such compounds into trials and make the UK less competitive as a place to perform clinical developments. Companies may move this work to other EU Member States, India or the Far East. And there's likely to be a shortage of healthy young volunteers for Phase I — they may think twice before going down this road to earn a bit of extra cash. The impact on manufacturing remains to be seen, but requirements for better characterization and more comparability data are likely to add to timelines and costs. It would be a pity if these constraints put a block on the development and manufacture of what could be important new therapies.  

Back to the patients. Prompt action by medical staff at Northwick Park thankfully saved the lives of the six young men affected by TGN1412's cytokine storm. But two have been left with long-term disabilities and all have ongoing damage to their immune systems, which is likely to have consequences to their future health. It is up to biotech companies and the regulatory authorities to make sure that this human tragedy never happens again.  

Last year, a monoclonal antibody, TGN1412, led to potentially fatal adverse effects in a small group of Phase I volunteers in London. In the wake of this incident, EMEA has drawn up new guidelines that could lead to demands for more data on novel biologics. They may have implications for both manufacturing and clinical trials of biopharmaceuticals that are considered to pose a high risk to patients.

Safety First

Large-scale protein production is not easy. You may have performed extensive work on host cell development, media selection and scale-up to develop a manufacturing system that can produce, say, as much as 20 g/L of high-value product to service clinical trial supply or even commercial demand. That is where the challenge — or fun, depending on your perspective — really begins. How do you turn thousands of litres of messy cell culture brew into vials of fluffy white protein powder, while under pressure to cut costs, timelines and meet regulatory demands on purity?  

While biotech clinical trial results often hit the headlines, the work that goes into purifying these drugs (so they can get into the clinic) is too often undervalued and underestimated. Yet purification can account for 30–50% of total manufacturing costs — for this reason alone, it deserves more attention.  

"If you are in purification, the major new challenge is matching the production capacity to the potential increase in output from the fermenters," says Tony Orchard of Pall, a major supplier of filtration and separation products to the biopharmaceutical industry. "The other challenges of process validation, meeting timelines for new products and plant flexibility at reasonable cost are just as big." 

The nature of the protein and its production system tend to dictate purification requirements. Put simply, recombinant proteins tend to be produced in microbial hosts, monoclonal antibodies in mammalian cell culture. In the former, you may need to worry about separating the product from cell debris and endotoxin contaminant, in the latter about inactivating viruses that may have the potential to infect patients.  

John Liddell, purification R&D manager at UK contract biologics manufacturer Avecia explains that the initial steps in purification from a microbial host depend upon whether or not the product remains within the cell, or is secreted, when fermentation is complete. "The aim is to have the target protein in solution for the start of chromatographic purification," he says. Therefore, intracellular protein products will need to be released by some form of cell disruption, such as high-pressure homogenization. Solid–liquid separation by either centrifugation or microfiltration then removes cell debris.  

The next stage is multistep (two to five steps) chromatography using orthogonal chemistries. Process development involves making each chromatographic step compatible with the previous one so product can be seamlessly loaded from one column to the next. According to a review conducted by Avecia, the most common first chromatography step is ion exchange, accounting for 75% of processes, with the second step most often being hydrophobic interaction chromatography, accounting for 60% of processes.  

Although ion exchange is very useful as the first step of purification chromatography because of its high capacity, which soon reduces high volumes, Liddell argues that mixed mode media are starting to come into their own. However, mixed mode can present more process development challenges because it has more adjustable parameters to consider. There are many other new chromatographic media coming onto the market — monolithic media, beaded media with different chemistries, for instance, and media with larger pore sizes for purifying pegylated proteins (products linked to a polymer chain, which give a longer lifetime in the body).  

He also notes an increasing trend towards high-throughput technologies in process development; for example, using robotic handlers with miniature columns or 96-well plates with mass spectrometry. Avecia has been evaluating the application of high-throughput approaches in overcoming some of the purification bottlenecks.  

Liddell also notes a trend towards the increased use of modelling and data mining techniques in purification. Proteins are notoriously fickle and it is still impossible to predict behaviour and characteristics from sequence. But misfolding, aggregation and proteolytic instability in a protein molecule can cause real headaches in purification and it is better to be forewarned, if at all possible. "Developments in structural biology will allow more and more prediction of purification relevant information from sequence," Liddell predicts. "More initial purification development is likely to be performed through modelling prior to starting experimental studies."  

There is also increasing interest in data mining, for individual companies and contract manufacturers, he adds. Avecia has information on more than 35 biologic production processes, which can be used to inform future processes.  

There are alternatives to chromatography for large-scale purification of proteins from both microbial and mammalian host cells, such as precipitation and crystallization. Recent reports from some companies suggest there is some cautious experimentation being conducted with these approaches. However, there is an understandable reluctance to abandon tried and tested chromatographic cascades that are approved by the regulatory bodies unless driven to by the demand of increased process volumes.  

However, such radical change may prove necessary. Most of the growth in biotech production is currently focused on monoclonal antibodies, with approximately 160 of these protein molecules in development. A monoclonal is typically captured on a protein A column in initial purification. This step, along with viral inactivation, accounts for a major part of the cost of purification. With antibody titres increasing, there is also concern regarding whether protein A is beginning to approach its capacity. Therefore, for cost and technical reasons, finding replacements for protein A is becoming something of a pre-occupation among manufacturers. There is no obvious solution, but some alternatives were summarized by Duncan Low, scientific director of process development at Amgen, at a recent meeting. These include bacterial immunoglobulin G binding protein, lectins, immunoaffinity columns, single domain antibodies (highly selective and the closest to protein A) and synthetic ligands. Less complex ligands are cheaper, but tend to be less selective towards the target.  

"Assuming that monoclonals continue to dominate and grow in demand, there will be some trade-off between the resistance to change from proven production platforms and the need for more drastic changes driven by volumes, costs, opportunity and competition," predicts Orchard.  

Getting from a cell culture to a purified biotech product is a demanding exercise involving many operations. Increasing productivity in the upstream part of biotech production is placing new demands on the purification process, which may lead to adopting new technologies.

Protein purification rules

A data package of nuclear magnetic resonance, infrared, ultraviolet and mass spectrometry (MS), plus elemental analysis, is the minimum you need to characterize a small molecule. It should be enough for publication if you are an academic, but if your compound is going into patients, you will need much more than this to satisfy the regulatory authorities. If the compound is a biopharmaceutical drug, then characterization becomes even more difficult because of the size of the molecules involved — be they recombinant proteins, monoclonal antibodies or DNA vaccines. Characterization is now one of the challenges faced by companies embarking on the manufacture of biosimilar drugs, as they must prove that their product has at least similar physical and chemical properties to the original. 

Unlike most small molecules, proteins can change their structure and composition depending on conditions, so characterization must take this into account. A change in pH or temperature may make a protein drug denature, or form a complex, or even a high molecular weight aggregate with other protein molecules. 

Changes in manufacturing conditions can lead to new patterns of glycosylation — the addition of sugar molecules to certain amino acid residues — which can alter the molecular weight and biological properties of the protein. A range of analytical technologies is required to capture these complexities, and there is still plenty of room for innovation.  

Proteins typically have molecular masses of several thousand Daltons, which used to be way beyond the scope of MS. However, this is a technology that has increased its power, while decreasing in price. The mass spectrometer can now accurately determine protein masses to within 1 Da. Given that most proteins have a molecular mass of several thousand Daltons, this is an impressive development, and MS is now the 'gold standard' in protein identification. 

But the mass of a protein cannot determine whether it has the correct three-dimensional (3D) structure, the one that will have the required biological activity. In biotech production, the host cell — be it microbial or mammalian — can generally be relied upon to produce the right amino acid sequence of the desired protein, but it cannot always follow through with folding it into the right shape. This is a particular problem with microbial production; mammalian cell hosts have more 'machinery' with which to perform post-translational modifications — that is, folding and glycosylation. Techniques such as light scattering, ultraviolet and visible spectroscopy, and circular dichroism can provide useful information on whether or not the protein has been folded correctly during its manufacture. Instruments, such as the Malvern Zetasizer (Malvern Instruments Ltd, UK), use light scattering to determine not just the size of a protein, but also its oligomeric state — monomer, dimer or more complex aggregate — in solution.  

Determining 3D structure, where each atom of a molecule is located in space, goes beyond knowing a molecular formula and is the only way of gaining insight into a small molecule or a biologic drug's pharmacological activity. X-ray crystallography (XRC) is another technology that has undergone amazing developments, with robotic handling allowing for routine, high throughput applications. However, the 3D structure of a protein in solution, where it is physiologically active as a drug, could differ from its structure in the solid state. And, although there are thousands of known protein structures from XRC in databases, some important proteins, such as the membrane proteins, cannot readily be crystallized. Efforts to understand and predict their structures depend upon mathematical methods and computer modelling, and these are still far more demanding than predicting small molecule structures.  

Changes in glycosylation, host cell protein content or folding from batch to batch are a particular problem in biotech manufacturing. But conventional manufacturing is not immune to dramatic changes in product with slight changes in manufacturing conditions. A drug may alter its polymorphic form; that is, the way the molecules are packed together in the crystal. The tale is probably well known enough by now, but when Abbott's HIV drug ritonavir began to precipitate out of its semisolid capsule formulation, the company's scientists learned that the drug molecule had changed its polymorphic form. The new form of ritonavir, induced by some slight change in manufacturing conditions, had different and less favourable properties compared with the form that had originally been marketed. Abbott was forced to find a way back to the original form, which it did. The result is that both companies and the regulatory authorities pay far more attention to the 'supramolecular' structure of small molecule drugs, which requires extra solid state analytical work with XRC and other techniques. In other words, looking at large-scale structures is as important for small molecules as it is for biologic drugs. 

One urgent requirement for characterizing biologic drugs is the development of high quality reference materials to which a product can be compared, and there are many efforts being devoted to this goal. For instance, the UK's National Institute for Biological Standards and Controls states that its mission is to safeguard public health by helping ensure the quality of biological medicines. At the heart of this work is the preparation, storage and distribution of World Health Organization International Standards and Reference Materials, to provide benchmarks for product quality. This is a vital contribution, but as biotech medicines become more complex, the analytical scientists will have to work hard to keep up with the demand for references.  

Meanwhile, the biotech industry could make characterization of its products easier by paying more attention to downstream processing and purification issues, creating a cleaner product that is easier to identify. Advances in affinity chromatography, as described by Professor Chris Lowe of the Institute of Biotechnology at Cambridge University at the recent BioProcess International conference, look promising in this respect. This innovative approach involves the application of molecular modelling to design and optimize ligands that are carefully matched to a particular biopharmaceutical product. When they are attached to a column, they can capture the product with a very high specificity so that when it is eluted it should be much easier to characterize.  

If the characterization issue is hard for small molecules and proteins, then it is even more difficult for more advanced therapies, for example, stem cells. Companies, such as Dynal/Invitrogen, are developing technologies for flagging up marker proteins on the surfaces of stem cells, and sorting them into purified populations. This is a world away from passing a small molecule drug down a column, but as medicines become increasingly complex, advanced analytical technologies are needed to characterize them.  

... the biotech industry could make characterization of its products easier by paying more attention to downstream processing and purification issues, creating a cleaner product that is easier to identify.