The market for monoclonal antibody therapeutics was approximately $24 billion (€15.2 billion) in 2007 with an estimated growth potential of up to $47 billion (€29.8 billion) by 2012.¹There are 21 marketed antibody products, five of which continue to dominate the market (Avastin [Genentech], Herceptin [Genentech], Humira [Abbott Laboratories], Remicade [Centocor Pharmaceuticals] and Rituxan [Genentech]) and collectively account for approximately 80% of the market share. There has been a notable swing towards biologics in pharma and biotech company development pipelinesviaa shift in strategic development or acquisitions. One of the more notable acquisitions in this area is the incorporation of Cambridge Antibody Technology (CAT), now MedImmune, by AstraZeneca in a deal worth £567 million (€718.3 million) in 2006. Monoclonal antibodies now represent the strongest growth area in the therapeutic proteins market sector. By 2009, it is forecast that they will account for 48% of all sales of therapeutic proteins.

The increased demand for therapeutic antibody productsper se, coupled with the need to administer most of these molecules at relatively high doses, translates into a manufacturing requirement for possibly hundreds of kilos of product collectively per year. Accordingly, optimizing the production efficiency of these molecules is an area of intense research within the CMO and biopharmaceutical community. This is particularly so where the cost of goods weighs heavily in the business models of innovator companies seeking to develop new molecules for less common clinical indications and the emerging sector for 'biogeneric' and 'follow-on' biologics.

One response to the demand is a shift to larger bioreactor size with, in some instances, bioreactors of 20000 L working volume. There has also been a move from continuous process operations to fed-batch processes, and an area of increasing interest is the optimization of the expression cell line itself. Here, there is focus on identifying and developing ways in which the genes for the protein of interest can be best incorporated into the host cell to give stable and consistent high levels of secreted and active protein. It is recognized that optimized high titre expression clones have significant commercial benefits dictating bioreactor size and the overall manufacturing cost of the therapeutic.

Maximizing biologics expression in mammalian cell systems

The vast majority (~60–70%) of antibody and recombinant protein molecules are produced in mammalian cells.²This reflects the basic fact that most biologics are glycoproteins and require at least a mammalian pattern of glycosylation to achieve satisfactory clinical efficacy. Mammalian expression cell lines produce an acceptable glycosylation profile so cell lines such as Chinese Hamster ovary (CHO) have traditionally been used to produce the majority of protein therapeutics. Other mammalian cells used include mouse SP2/0 and, more recently, proprietary human retina-derived cells PER.C6. Further development of the CHO cell lineviathe introduction of human glucosaminyltransferase and fucosyltransferase genes aims to produce a more human-like pattern of glycosylation. Such 'glyco engineering' strategies are also being applied to nonmammalian host cells, such as yeast. It remains to be seen whether these and other nonrodent cell production platforms, including plant cells, transgenic animals and transgenic plant production, will begin to replace traditional platforms.

The pattern of glycosylation is particularly important regarding biogeneric molecules where 'bioequivalence' and being able to demonstrate identical or near similarity to a previously marketed reference biologic are critical to obtaining market approval. Technological developments such as the lectin array technology of Procognia (Israel) may surpass existing HPLC-based methodology in being able to rapidly monitor and define the glycochemistry of biogeneric and other glycoproteins during the development and production cycle.

Unlike bacterial expression systems, mammalian cell culture systems have a number of constraints such as relatively low protein yield and a lengthy development time required to select stable expression clones with optimal manufacturing profiles. To overcome these limitations, companies have placed a priority on optimizing the cellular expression systems used to produce the therapeutic. For example, Boehringer Ingelheim (Germany) through its BI HEX process has optimized its approach to working with CHO cells to give high titre expression in animal component-free media, and can claim productivity of >50 pg per cell per day (pcd) and in excess of 6 g/L in fed-batch processes for antibody production.

Enabling the cell line to grow at a higher density in culture is an additional consideration when developing an optimized production protocol. The human cell line PER.C6, developed by Crucell (The Netherlands), can grow to a very high density in culture with high cell specific productivities of >50 pcd in some instances, translating to an excess of 5.8 g/L or even higher in a fed-batch environment. Compared with the traditional CHO system, a human cell line may exhibit more favourable post-translational processing of the therapeutic, but differences in sialic acid group derivatives in human and other mammalian proteins lead to the possibility of host immune cell responses (immunogenicity) evenviathis route of production.

A need to reduce development time has spurred the application of high-throughput automation to identify cells with the desired productivity and other phenotypic attributes. One example is the ClonePix FL instrument (Genetix Ltd, UK), which identifies and selects desirable clones. Similarly, a system employed at Pfizer (CN, USA), known as the 'workcell' robot, is routinely used to rank and select suitable clones prior to scale-up in bioreactors. In some groups, fluorescence-activated cell sorting machines have also been employed to screen and select relevant clones at the single cell level.