Research review paperUpstream processes in antibody production: Evaluation of critical parameters
Introduction
Monoclonal antibody (mAb) based therapeutics have become an important class of drugs and diagnostic agents, specifically for treatment of human malignancies and other chronic inflammatory conditions. This is much due to their specificity and selectivity that their demands have risen in the last few years (Reichert et al., 2005, Chu and Robinson, 2001, Adams and Weiner, 2005). The original method for production of monoclonal antibodies involved use of murine hybridoma cells. Therapeutic murine antibodies were made commercially available since 1980, but could not gain much success as they instigated immunogenic reaction on administration and thus were rapidly cleared from the body. Attempts were made to improve the efficacy of the antibodies using two different strategies; one involving production of chimeric monoclonal antibodies (mAbs) in which the Fc portion of the murine mAbs was replaced by human Fc while the other was to produce fully human mAbs (Morrison et al., 1984, Boulianne et al., 1984, Cole et al., 1984). Reichert et al. (2005) have analysed the current trends in mAbs production and reports an inclination towards study of human mAbs and mAb fragments. The data collected by them indicates FDA approval rates of chimeric and human mAbs are in the range of 18 to 29%. Eighteen mAbs have been approved for therapeutic use out of which three are murine antibodies and produced in hybridoma cells, others are chimeric, human antibodies (Chu and Robinson, 2001). Hundreds of other mAbs are in different phases of clinical trials. The global market for mAbs is expanding rapidly and in a recent analysis by Tufts center for study and research for drug development, Tufts projects that by 2008, the U.S. Food and Drug Administration is likely to approve 11 of the current mAb products in the pipeline and total market is going to reach US$ 16.7 billion (Pavlou and Belsey, 2005). In effect, these approvals would come close to doubling the number of mAbs now on the market. According to the report, newer mAbs, which include humanized mAbs and human products, are of types that have had a much higher success rate in attaining FDA approval than original murine (mouse derived) products. Historically, chimeric mAbs (such as the recently approved cetuximab [Erbitux]) have had the greatest success with FDA approval, with 26% of products in development hitting the market (Reichert and Pavlou, 2004). The approval rate for murine products, by comparison, is 4.5%. The current global market share of modified and engineered antibodies is around 56% (in 2006) and which is likely to remain 54% by 2011 as estimated by BCC research reports. This rapid increase in the number of mAbs being regularly approved for therapeutic use puts up a need to produce sufficient quantity of these mAbs which require sophisticated but commercially simple process operation. This clearly indicates a requirement to develop such bioreactors or processes which can be scaled-up easily and also produce mAbs in grams.
Antibodies are amongst one of the most expensive therapeutics being used, this is mainly due to their use for chronic diseases in high dose (≥ 100 mg) (due to their low potency). Consequently large scale processes are required to produce 10–100 kg/year to cope up with market demands. The upstream processes have been significantly improved (Birch and Racher, 2006, Farid, 2007). With respect to production systems major improvements have been made in the cell lines and production systems being used for production of antibodies. Depending on the specific use, different forms and different production hosts might be desirable and several recent reviews have specifically addressed issues surrounding production in microbial systems (Humphreys and Glover, 2001, Humphreys, 2003) and transgenic plants (Hood et al., 2002), as well as general production issues (Roque et al., 2004).
In particular for production of antibody fragments microbial system has gained considerable importance and E. coli cells are widely used for this purpose. A variety of approaches have been adopted by different groups to improve the production levels of antibody fragments using the E. coli system. Expression levels of up to 2 g/L have been reported (Chen et al., 2004). Recently, Mazor et al. (2007) have reported isolation of full length genetically engineered IgG antibodies by using combinatorial libraries of E. coli. In spite of all this, mammalian cells remains the best choice for the production of antibodies. This is mainly due to their capacity for proper protein folding, assembly and post translational modification. Thus the quality and efficacy can be superior compared to other host systems such as bacteria, plants and yeast (Wurm, 2004, Andersen and Reilly, 2004). Starting from production of monoclonal antibodies in murine hybridomas, progress has been made to produce human monoclonal antibodies and a number of genetically engineered cell lines have been used for this purpose. The cell lines which are currently being used include CHO, NS0 and Sp2/0-Ag 14, and hybridoma cell lines (Chu and Robinson, 2001, Wurm, 2004, Andersen and Reilly, 2004).
Hybridoma cells are produced by fusion of a myeloma cell line with B cells i.e., the antibody producing cells (Fig. 1). Initially murine cells were used for this purpose. Now in addition to murine antibodies, it is possible to make human antibodies using murine hybridoma technology. However, unlike the original hybridomas (Köhler and Milstein, 1975), the spleen cells are taken from a transgenic mouse which has the murine immunoglobulin locus replaced by the human genes. The productivity of these cell lines have been improved greatly by genetic engineering and different expression vectors have been incorporated in these cell lines which can give high specific production rates (Qp, = 90 pg/cell/day) (Wurm, 2004, Birch and Racher, 2006). The two most commonly used systems for the production of therapeutic monoclonal antibodies are the glutamine synthetase (GS) gene expression system (Lonza Biologics; (Birch et al., 2005)) and those based on dihydrofolate reductase (DHFR) genes.
Apart from the advancements in the host systems major increase in the volumetric productivity have been achieved due to media optimizations and process control (Wurm, 2004, Birch and Racher, 2006). Control of biological processes is a complex operation due to non-linear nature of these processes. The response of biological systems to any change in environment or its process is less reproducible which is a major limitation while development, optimization and scale-up. Different control parameters which characterize a basic chemical engineering process are not so easy to control and operate in biological systems (Sommerfeld and Strube, 2005).
Process optimization involves optimization of a chosen set of values of the variables subject to the various constraints that will produce the desired optimal response for the chosen objective function. Almost all the process optimization techniques described in the literature aims at increasing the culture longevity and thus increasing specific antibody production rate and overall total antibody yield at the end of the process. Thus with increase in the demand for such therapeutically important recombinant protein products like monoclonal antibodies, the production process has been technically improved both at upstream and downstream level. The processes have been scaled up from 500 L in early nineties to a production capacity of 20,000 L till date (Lonza Biologics).
This review focuses on the techniques used for production of monoclonal antibody and the major advancements made in the upstream processes henceforth. The upstream processes parameters generally involves designing of new bioreactors or modifying the existing ones, or optimizing the existing processes, besides manipulations with the culture system and medium optimizations and this review aims to highlight some of these process units, their advantages and limitations.
Section snippets
Bioreactors for antibody production
In vitro cell cultivation techniques play a key role in production of therapeutics like proteins, antibody, diagnostics, vaccines etc, and for tissue engineering applications (Martin et al., 2004, Martin and Vermette, 2005, Wang et al., 2005, Warnock and Al-Rubeai, 2006). Bioreactors are the base where in vitro cell cultivation could be done under strictly controlled conditions and can be monitored regularly (Fig. 2). The field is regularly advancing and many new bioreactor designs and
Process optimization for antibody production
Persistent efforts by researchers have led to better insights into cell physiology and metabolism, making it practically feasible to identify and anticipate various process parameters which affect productivity of the system. This has led to testing and development of mathematical models and online measurement instruments for process control. Optimization of the process, online monitoring and prognosis of the outcome leads to better control and reproducibility of the system, which is otherwise
Conclusion
The therapeutic importance of monoclonal antibodies has led to increased demand which makes it worthwhile to expand the existing facilities for its manufacture in terms of scale. Upgrading the prevailing technology in terms of efficiency and economy is one way to cope-up with the increasing demand. On the other hand to test the production at initial stages and to speed-up the process for getting faster approvals, disposable bioreactors and other disposables at downstream level are becoming
Acknowledgements
The work was financially supported from the grants from Indian Institute of Technology, Kanpur, India and Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India. EJ gratefully acknowledges the fellowships received from Indian Institute of Technology, Kanpur, India for her Ph.D program.
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