Biologics build a better base

Sarah Houlton reviews the state of biologics and discovers that while there are still many difficulties to overcome, the use of large molecules in treating disease is becoming more commonplace.

The biotech revolution has led to increasing numbers of biological medicines reaching the market. These are usually complex, large molecules, which cannot be made easily by traditional chemical synthesis routes, meaning that biological processes have to be used to make them instead - a very different proposition from standard small molecule manufacture.The earliest biologics, such as the growth hormone EPO, were needed only in small volumes. However, biologic medicines to treat common chronic diseases like arthritis are today coming on stream, not to mention products that need to be administered in larger doses, meaning that the ability to manufacture at bigger scales is becoming more and more essential.

long incubation

Many biologics are made using mammalian cell culture technology, which uses recombinant DNA technology to introduce relevant genes into cells. These cells are then used to make the desired protein, which is ultimately fished out of the complex 'soup' at the end of an incubation period lasting weeks or even months.

The first stage of the process is development of the cell line: this involves the gene that codes for the desired therapeutic protein being inserted into the genome of the host's cell. The recombinant cells that perform best are then selected, and can be frozen in ampoules for preservation purposes. Storage in a cell bank under liquid nitrogen at temperatures as low as -180°C is possible.

The 'business end' of the process takes place in the bioreactors, which can range in size from small glass bottles to huge reactors with a capacity of thousands of litres. Firstly, an ampoule of cells is taken from the cell bank and its contents are thawed. The cells are then multiplied in a suitable culture medium, and once there are sufficient cells they are put in the bio-reactor and fed with a nutritious culture medium containing substances such as salts, sugar, vitamins, and air to provide oxygen.

In the reactor the cells will make a number of different proteins, including the desired therapeutic protein. The state of the cell culture and the quality of the product are regularly checked, and the mix containing the protein is then harvested and separated from the cells for purification: this involves several consecutive steps, beginning with filtration.

Typically, ultrafiltration will be used to concentrate what comes out of the reactor to around a tenth of its original volume, and will be followed by chromatography to separate different molecules. Various different forms of chemistry are used, including size exclusion, which acts like a sieve, allowing proteins to pass through a resin at various speeds according to their size, with the smallest molecules coming out first.

fed-batch technology

There are essentially two reactor technologies that are used in mammalian cell culture: the older perfusion technique, and the fed-batch technique that has been developed more recently. In a perfusion bioreactor, the incubation process will take two or three months, with the culture medium being gradually fed into the bioreactor over this time. In the newer fed-batch technique, the medium is fed to the bioreactor over a shorter period, typically three weeks, meaning that the complete harvest is taken in one batch, with the entire reactor contents being separated using a centrifuge before purification. Furthermore, perfusion reactors are usually up to 300 litres in size, whereas fed-batch bioreactors are better adapted to larger scale production requirements.

major investment

One company that has been making significant investments in mammalian cell culture manufacturing is Serono. The Swiss-based firm is the world's third biggest and Europe's largest, biotech company. Its current major product, multiple sclerosis treatment Rebif (betaseron), reached blockbuster status last year, with sales topping US$1bn. It has been on the market since March 2002, when it gained approval for the treatment of relapsing forms of MS, having overcome a competitor's orphan drug status based on superior efficacy in relapse reduction.

Serono has 10 manufacturing sites, including the Serono Biotech Centre (SBC) at Corsier-sur-Vevey, near Lake Geneva in Switzerland, where it is making substantial investments; several 5,000-litre fed-batch reactors are already in place, and plans are afoot to install a further 12 with a capacity of 15,000 litres each. The expansion has become necessary because of the increasingly widespread move away from small-dose hormone type products towards newer large-dose therapeutic proteins and monoclonal antibodies.

When the facility, on the site of an old tobacco factory, was first developed, it was designed to allow space for expansion. Serono began making Rebif there in 2000, and it is now implementing a dual sourcing back-up strategy with the aim that all of its products will be manufactured at more than one site to ensure continuity of supply in the event of a problem at any of the sites.

expansion plans

'We have dozens of bioreactors at the SBC,' said the company's senior vice president of manufacturing, Michele Antonelli. 'These range in size from 50 to 5000 litres. Some processes are still in roller bottles, where the process works well. We are looking to expand our bioreactor capacity to a total of 200,000 litres.'

Jonathan Barnsley, site director of the Vevey plant, said: 'when we opened the SBC only one third was developed; this means that we are able to respond rapidly to market needs.' Even after recent capacity additions, a third of the building is still available for the introduction of the planned 15,000 litre reactors.

'The molecules we are working with now have to be administered in larger quantities, so we are making them on a larger scale. Processes only work if they can be scaled up to a large scale,' added Antonelli. 'The need to minimise immunogenic reactions makes product purity very important in the development of new processes. Production costs also have to be worked on at an early stage because it has to be bearable and the products have to be acceptable to patients.'

The SBC also has four 300-litre perfusor bioreactors, while Rebif is made using a 75-litre perfusor. Some products can only be made using these types of reactors, especially if the products are very unstable with temperature or time; for example, if they begin to degrade when they warm up. At any one time the company is normally running between six and eight of these perfusor bioreactors.

Mammalian cell culture makes a high quantity of very dilute product, while even the best protein manufacturing processes will make no more than around 7g per litre, with most making much less. The practical aim is to get a few kilograms of product from a few thousand litres of reactor. Interferon is a substance made by cells in trouble, and Rebif is a very low concentration product - not even grams a litre - meaning that large volumes are required to manufacture sufficient amounts for clinical needs.

purity issues

As well as production at the SBC, the company carries out development work, working in cell sciences and the improvement of cell culture media, and investigating upstream and downstream processes, such as better purification methods.

Purity is a big issue for therapeutic proteins, with the byproducts including incomplete protein chains and components of the culture media. Another problem is that the product being made can interfere with the cells, thereby slowing down the reaction and negatively affecting cell yield.

A further time-consuming issue is the need for cleaning and validation of the reactors between batches; this is why Serono is moving towards the predominant use of disposables. Throwing away a plastic bag liner is much quicker than cleaning one, and such disposability will help to make the process more flexible.

Increasing production capacity is not just a case of installing more, and larger reactors. Doubling the productivity of the cells will double the amount of product that can be achieved without doubling the reactor size. 'It's more efficient to get the cells to improve the process,' said Barnsley.

'If you can reduce cycle times by two days per run, this will make a big efficiency saving over the year,' pointed out Tim Clayton, manager of technical support, SBC. 'A good process will make the most of a cell line. However, once the product is licensed, if the process is sufficiently altered the product will also be altered, possibly resulting in the need for new clinical trials. Advantages can come from keeping the cells alive for longer.'

It is essential to ensure that the product is as pure as possible, as is proving that there is very little variability from batch to batch; something that is not as simple to ensure in large molecule chemical manufacture as in small molecule chemical manufacture. This is because there are more factors that can influence the production process, such as the cell line, the media used and the temperature; making careful checking essential to keep the reaction process under control.

'We sample at all the stages of the process,' explains Barnsley. 'The samples are analysed in quality control labs to ensure they comply with the regulatory authorities' specifications.'

Advances in genetics research mean that medicines like therapeutic proteins are only going to increase in importance in the future, and as more products reach the market, treating chronic conditions from arthritis to heart disease, it is going to become ever more important to have reliable, large scale manufacturing processes. Cell lines will be made more efficient, and culture media that encourage the cells to make more of the required proteins will be developed as biotechnological manufacturing methods become as routine as small molecule chemical synthesis is today.