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Enzyme substitutions involving recombinant DNA technology

Worldwide, less than 10,000 people suffer from Gaucher’s disease, which is the most common lysosomal storage disease. Genzyme has been offering the drug Cerezyme® for the treatment of this rare, genetic disease since 1994. The company’s subsidiary in Constance markets and sells this enzyme replacement therapy in Europe and Asia. The active substance is produced in Chinese hamster ovary cell cultures.

Gaucher’s disease, which was described for the first time by the French doctor Philippe Charles Ernest Gaucher in 1882, is an autosomal recessive disease caused by a defect in the lysosomal enzyme glucocerebrosidase. Glucocerebrosidase cleaves glucocerebrosides into glucose and ceramides. Glucocerebrosides are generated during the degradation of the membranes of dead white and red blood cells and, in healthy people, are degraded in the lysosomes of macrophages by glucocerebrosidase. Gaucher’s disease sufferers lack this enzyme, and accumulate glucocerebrosides in ‘Gaucher storage cells’. “The fatty material collects mainly in the spleen, liver, kidneys, lungs, brain, bone marrow and lymph nodes and leads to a broad range of different disease symptoms,” said Dr. Michael Preiss, Medical Director of Genzyme CEE GmbH in Constance.
Gaucher cells accumulate mainly in spleen and liver. (Photo: Genzyme Corporation)
Gaucher cells accumulate mainly in spleen and liver. (Photo: Genzyme Corporation)

Slight to very severe symptoms

Disease symptoms include the up to 60-fold enlargement of the spleen, the 10-fold enlargement of the liver associated with distinctive abdominal problems, breathing impairment and painful bone symptoms and bone infarctions. The Gaucher cells displace the bone marrow as they grow and degrade the bone substance, which leads to deformations and spontaneous fractures. Infarctions and knot formation in the organs lead to the shrinkage and function loss of the affected organ in later stages of the disease. In addition, Gaucher’s disease patients suffer from increased susceptibility to infections, anaemia, blood coagulation disorders and an elevated risk of cancerous diseases. Severe cases of Gaucher’s can lead to death. Without treatment, patients usually die on average 53 years after the disease was diagnosed, mainly due to organ manifestation and malignant tumours. Patients suffering from Gaucher’s diseases that also affect the central nervous system have a much shorter life expectancy.

Gaucher’s disease has a worldwide incidence of 1:100,000. It is an autosomal recessive disease, which means that affected individuals must have inherited two mutated copies of the glucocerebrosidase gene (one from each parent) in order for the disease to develop. A person with one defective gene and one normal glucocerebrosidase gene is a carrier of the disease without actually developing the disease as the normal gene produces sufficiently high amounts of enzyme and is hence able to compensate the defective gene. Carriers have a 50:50 chance of passing the Gaucher gene on to their children. Experts classify Gaucher’s disease into different types depending on the onset of disease, the symptoms and the involvement of the central nervous system. The disease has three clinical subtypes: type 1 (nonneuronopathic type), type 2 (neuronopathic type) and the rather rare type 3 (chronic neuronopathic type), characterised by gradual mental prostration, CNS complications and seizures.

Hamster cells for reliable enzyme production

Managing director of Genzyme CEE GmbH: Dr. Ute Stölzle (Photo: Genzyme)
Genzyme, founded in Cambridge, MA, USA in 1981, started very early with the development of drugs for the treatment of Gaucher’s disease. “The first medication that was able to replace glucocerebrosidase was Ceredase, a macrophage-specific placental glucocerebrosidase enzyme,” said Dr. Michael Preiss. Cerezyme®, a drug which later replaced Ceredase, is an enzyme substitution therapy that received marketing authorisation from the American FDA in 1994 and from the European Commission in 1997. “While Ceredase was still produced from human cells, recombinant Cerezyme® is produced “biotechnologically in Chinese hamster ovary cells (CHO cells), which produce human glucocerebrosidase,” said Dr. Michael Preiss. The method is well established and is now standard for the biotechnological production of vaccines, antibodies and hormones. Enzyme substitutes for lysosomal storage diseases such as Fabry disease, mucopolysaccharidosis type I and Pompe disease are also produced with CHO cell lines. A big advantage of CHO cells is the low risk of human viruses being present, in contrast to human cell lines used for the production of the required enzymes.

The production process starts with the isolation of the human glucocerebrosidase gene, which is then integrated into a transport DNA (plasmid) and introduced into CHO host cells. These cells offer optimal preconditions for the production of the desired protein. Under controlled conditions, the CHO cells secrete glucocerebrosidase into the culture medium. A multi-step purification process follows during which the human gene is isolated and modified in several steps. Carrier substances are subsequently added and the drug is freeze-dried. The process ends with the final control of each of the Cerezyme® units, which involves 30 steps. Cerezyme® is administered intravenously and reaches the target cells by way of the blood. Patients often report considerable improvements within a few weeks of the first treatment. Pathogenic blood disorders (anaemia, leukopenia) as well as enlarged livers or spleens improve considerably. During further treatment with Cerezyme®, skeletal disorders and lesions improve. The patients have to take the enzyme substitute for life, the dose is adapted to body weight and therapeutic success.

Further development for more therapeutic options

Dr. Michael Preiss told us that Genzyme has established a comprehensive small molecule research and development programme in the area of lysosomal storage diseases. Genzyme is working hard to develop an oral medication (capsule) for the treatment of Gaucher’s disease. “The Constance-based company has been selling four drugs for the treatment of genetic diseases in 20 Eastern European countries (Balkan, Russia, Ukraine, and others) since 1995,” said Dr. Ute Stölzle, Managing Director of Genzyme CEE in Constance. The costs for one-year’s treatment using these enzyme substitutes are very high due to enormous research and development costs and low patient numbers. Depending on body weight and drug dose, the costs for a one-year enzyme therapy amounts to between 50,000 and 300,000 euros. Genzyme undertakes a number of humanitarian support programmes for patients that are unable to afford the vital therapy for economic or country-specific reasons. These patients receive the drug free of charge. About 40 per cent of all people treated in Europe and Asia receive Genzyme products such as Cerezyme® free of charge.

Ute Stölzle states that Genzyme does not achieve any commercial benefit from this, and says that the company has to develop its market position in the same way as any other company. “However, the orphan drug legislation encourages companies to develop drugs for rare diseases,” said Stölzle. For example, research-based biotech companies are supported by institutions such as the FDA’s Office of Orphan Products Development (OOPD) in the clinical testing of new drugs.

mst – 18 August 2008
© BIOPRO Baden-Württemberg GmbH

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