MaBnet

MabNet A strategic Canadian program to develop production technologies for monoclonal antibodies

MabNet student growing EG2 cells in a bioreactor

Introduction

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.

Manufacturing

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.

Glycosylation

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,

HUMAN AND LLAMA ANTIBODIES

fucose-free Mabs can be produced by various means including genetic inactivation of the fucosyl transferase gene or by the use of specifi c inhibitors in the culture.

MabNet

The objective of MabNet is to understand the functional properties of specifi c Mab glycoforms and to be able to develop bioprocesses that enable the production of single glycoforms or at least a restricted glycoform profi le. An understanding of the function-structure relationship is key to developing Mabs with enhanced therapeutic properties. The control of glycosylation in the biomanufacturing process is essential at various levels. Firstly, it’s important to ensure consistency of products between different batch runs. Secondly, it’s important to enrich the fi nal Mab product with the glycoforms having the desired effect.

MabNet has chosen to study an unusual but potentially important therapeutic antibody that was derived originally from a llama. It turns out that all camelids (family of camels and llamas) have small antibodies that lack the usual light chains that are present in humans. The selected MabNet antibody is one targeted to epidermal growth factor receptor (EGFR) which is strongly expressed on specifi c cancer cells. The value of the small size of the camelid antibody is that it exhibits enhanced penetration of solid tissue, giving it good access to the cancer cells in vivo.

The selected clone of the MabNet antibody (labeled EG2) has been fused with a human constant region structure to make it less immunogenic and transfected into Chinese hamster ovary (CHO) cells. These are the most predominant cell line used for biopharmaceutical production because of their ability to synthesise proteins with a human-like structure, particularly with respect to the glycan profi le. Although the EG2 Mab has potential as a therapeutic in its own right, the focus of the MabNet program is to use this as a model antibody to develop platform systems that could be applicable to a wide selection of antibodies that require large-scale manufacture. The platforms developed by the network embrace all aspects of production including cell line development, bioanalytics, bioprocess control and downstream processing. These are organized into four themes related to the expertise of individual researchers. The value of this organization is that it cuts across the traditional subject boundaries so that there is strong interaction between different subject areas.

Up to now the MabNet has generated aberrant glycan profi les of the EG2 Mab by the use of specifi c glycosylation inhibitors, by mutagenesis and by enzymatic re-structuring. The structures have been determined by a combination of exoglycosidase array analysis, HPLC and mass spectrometry. Specifi c functional binding and cell-based assays will be performed on these structural variants to see the structure-to-function relationship. Bioprocesses are being designed based upon the highly successful fed-batch principles used in industry. This involves periodic feeding of nutrients to maintain an effi cient metabolism that maximizes cell viability during the later stages of the process. However, it has been shown that limited levels of carbon and nitrogen substrates can compromise glycosylation which can in turn reduce the quality of the fi nal purifi ed Mab. With this in mind, the bioprocess is being modeled by metabolic profi ling and fl ux analysis to ensure adequate control of the fed-batch cultures to maximize the production of high value Mabs. The downstream processing part of the MabNet program is directed to the need for the high level purifi cation necessary for the production of human injectable products. Here it is essential to ensure that the Mabs are maintained in their native and monomeric state as they are concentrated and formulated as clinical products. From a commercial perspective downstream processing has become particularly important because of the high costs involved relative to others parts of the bioprocess.

An equally important objective of the MabNet program is to train postgraduate students in the art of biomanufacturing so as to increase the pool of highly skilled personnel in an industry that is dependent on the availability of trained recruits. At the moment there are 32 graduate students associated with the MabNet program and scattered throughout the country. Co-ordination and communication is maintained regularly by teleconferences and formal presentations at conferences such as the biotechnology sector of the Canadian Society of Chemical Engineering. The network is also preparing a training conference in the fall of 2012 which will focus on the business and commercial aspects of biomanufacturing. The rationale is to ensure that the trained graduate students from the program have both the potential for good science as well as good business acumen. This will be a clear benefi t to the Canadian corporate sponsors of MabNet and it is hoped that this pool of graduates will help promote biomanufacturing as a major sector of the Canadian economy in the future.

References

1. Repligen. www.repligen.com. Annual

Report 2011. 2. Datamonitor. Monoclonal Antibodies; 2010. 3. Reichert JM. Which are the antibodies to watch in 2012? mAbs 2012; 4:1-3. 4. BCCResearch. Antibody drugs: technologies and global markets. Biotechnology 2012.

Michael Bulter is a Distinguished Professor at the Department of Microbiology, University of Manitoba, Winnipeg, Manitoba and Scientifi c Director of MabNet. He can be reached at butler@cc.umanitoba.ca.

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