By: Michael.Butler
MabNet
Monoclonal Antibodies
A strategic Canadian program to develop production technologies for monoclonal antibodies Glycosylation
MabNet student growing EG2 cells in a bioreactor
Introduction
Manufacturing
There has been a steady increase in the number and demand for biopharmaceuticals for the treatment of human diseases. Global sales of biologics are now reported at $120 billion per annum with an expected increase to $150 billion by 20151 and clearly outpace the growth of small molecule therapeutics. This economic success has been dominated by humanized monoclonal antibodies (Mabs) produced from mammalian cell culture bioprocesses. Despite the global economic downturn the annual growth rate for Mabs is predicted at 9.5 per cent up to 2015.2 They are the largest and fastest growing class of therapeutic pharmaceuticals with around 28 approved and around 350 in various stages of clinical trials.3 This growth is likely to continue because of their application to areas of unmet medical need and focus on novel targets which are associated with a reduced competitive intensity. The global sales of Mabs in 2011 were estimated at $44.6 billion and are predicted to increase to $58 billion by 2016.4 There are presently six blockbuster therapeutic Mab products on the market (greater than $1 billion annual sales): Avastin, Herceptin, Remicade, Rituxan, Humira and Erbitux, all of which have been highly successful in the treatment of specific conditions including rheumatoid arthritis and specific forms of cancer.
Although monoclonal antibodies have been around since the 1970s following the Nobel Prize-winning work of Kohler and Milstein on immortalization of B-lymphocytes, their full potential as therapeutic drugs was not developed until their structures were “humanized” in a form that would not lead to the immunogenic responses associated with mouse antibodies. The present high demand for Mabs as therapeutics brings into question the optimal methods of manufacture. This was a question addressed by a group of Canadian scientists from eight universities, 14 sponsoring companies and three government institutes. The multidisciplinary group of molecular biologists, biochemists, cell biologists and chemical engineers formed a network which has been funded by the manufacturing sector of NSERC to design optimal methods of production of selected antibodies. With the University of Manitoba as host institution, MabNet (www.mabnet.ca) is presently starting its third year of operation with a million dollar per annum operating budget from NSERC. This is supplemented by sponsorship and operating funds from 14 of the most prominent Canadian companies in biotechnology and biomanufacturing, who benefit from the scientific work arising from the network.
The major focus of the MabNet research is the optimization and control of Mab production from mammalian cells in culture. One particular aspect of this is the control of glycosylation of the Mab which relates to the carbohydrate structure associated with the protein part of the molecule. Although at one time the carbohydrate structure (also known as “glycan”) was thought to be an unimportant adornment of glycoproteins, there is considerable data to show that glycan variants of proteins affect biological activity. All antibodies have glycans at a conserved site on the Fc region of the protein. However, for each antibody there may be up to 30 different structural forms of the glycan. Alterations of the glycan profile of antibodies can be affected by the physiological and pathological conditions in vivo, particularly aging, pregnancy or disease. Decreased terminal galactosylation has been shown for patients with rheumatoid arthritis or several forms of autoimmune disease. Differential sialylation has been shown to affect the inflammatory properties of IgG and has been proposed as a mechanism of a molecular switch to induce an anti-inflammatory condition. It would seem that these alterations in activity are related to the differential interaction of the Fc domain of the IgG with a series of natural receptors. The glycan profile can be very important for therapeutic Mabs that require multifunctional activities for clinical efficacy. Mabs are complex glycoproteins with functions that include antigen-binding through the Fab variable domain and effector functions through the Fc constant domains. The glycosylation of Mabs is particularly important for the effector functions which are required for most therapeutic applications and may be improved by specific modifications of the glycan. For example recent work with the therapeutic antibody, Herceptin® for the treatment of breast cancer, has shown that a glycoform with no fucose has up to a 100fold higher binding capacity to the receptor that triggers its therapeutic activity, the so called antibody-dependent cell cytotoxicity (ADCC). The enhancement of this activity allows the Mab to be effective at lower doses. Although fucosylation is a normal activity during glycosylation in mammalian cells, MAY 2012 BIOTECHNOLOGY FOCUS 11
