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ANTIBODY DESIGN AND PRODUCTION

Submitted by- Rajkumar (80), co author - Sunny Tonk (107)

The clinical and commercial success of monoclonal antibodies has led to the need for very large-scale production in mammalian cell culture. This has resulted in rapid expansion of global manufacturing capacity [1], an increase in size of reactors (up to 20, 000 L) and a greatly increased effort to improve process efficiency with concomitant manufacturing cost reduction. This has been particularly successful in the upstream part of the process where productivity of cell cultures has improved 100 fold in the last 15 years. This success has resulted from improvements in expression technology and from process optimisation, especially the development of fed-batch cultures. In addition to improving process/cost efficiencies, a second key area has been reducing the time taken to develop processes and produce the first material required for clinical testing and proof-of-principle. Cell line creation is often the slowest step in this stage of process development.

1. Introduction

Currently, there are 18 monoclonal antibodies approved for therapeutic use [2]. The majority (15) of these antibodies are produced by recombinant DNA technology although three are murine anti- bodies made in hybridomas. The recombinant anti- bodies are produced in mammalian cell expression systems using Chinese hamster ovary (CHO) or murine lymphoid cell lines (e.g., NS0, Sp2/0-Ag14). Most products in clinical trial are whole antibodies made in mammalian cell systems but some are antibody fragments, which can be made in micro- organisms such as E. coli. For example, CIMZIAk [2] is a pegylated FabV fragment made in a microbial system and is currently in phase III trials. Reichert et al. [2] report that, of the 15 antibodies they identified in phase III trials, six were single chain or Fab fragments. In this article, we review the technologies used to manufacture antibodies focusing particularly on the current status of mammalian cell culture and approaches taken in process development. There are two crucial issues, which have to be faced in process development. The first is to minimise the time taken to provide material for clinical studies and the second is to develop a process which can deliver sufficient drug substance to meet market demands at an acceptable price per dose.
The industry continues to look at new technologies and process development strategies that will reduce timelines. The resulting processes must be easily scaleable, robust and meet quality and safety criteria. One approach to shortening the timelines is the use of platform technologies for cell culture processes, for example using standard media, feeds and growth conditions. Cell line construction and selection is often a critical path activity and needs to be completed rapidly without compromising quality criteria. Ideally, one would like to rapidly create a highly productive cell line that could be used for long-term manufacture obviating the need to create an improved second generation cell line at a later stage of development.
Productivity of mammalian cell processes has improved dramatically in recent years [3] and modern cell culture processes can achieve antibody concen- trations exceeding 5 g/L [4, 5]. This has resulted from improvements in expression technology and from process optimisation, particularly of the upstream, cell culture stage. Most current processes are based on fed- batch culture and the development of feeds in particular has made a significant contribution to increased antibody yields. Highly productive cell lines result from using a host cell line that has the desired characteristics, an appropriate expression system, and a good transfection and selection protocol. A number of expression systems with the potential to produce cell lines with high specific production rates ( Qp) are available. The challenge is to create cell lines that not only have high Qp but also have the growth characteristics that will lead to high productivity in the manufacturing process. This can be achieved by selecting cell lines with the combination of a high Qp, an ability to achieve high spacetime yields of viable biomass and consequent high volu- metric production rate in a screen designed to mimic the final production process. The resulting cell lines are also selected for stability, growth in suspension culture using a chemically defined, animal component free (CDACF) medium and the ability to make the desired post-translational modifications.
Purification strategies are based on chromatographic procedures usually including a protein A affinity step. With increasing upstream concentrations, significant attention is now being paid to improving downstream efficiency.

Reactor systems used for large-scale antibody production

A consequence of the rapidly growing demand for monoclonal antibodies has been a dramatic increase in capacity in the industry [1] and an increase in the scale of reactors used for production. Two types of culture system are used for large-scale manufacture, fed-batch and continuous perfusion culture [33]. The principles of these types of reactor are shown in Figs. 1 and 2. Fed-batch processes are by far the most common and are now operated at scales up to 20000 L working volume. Several authors [33, 34] have reviewed the types of cell culture reactor systems and processes in industrial use.

In fed-batch culture, small volumes (in our case less than 10% of the reactor volume) of key nutrients are fed to the culture during the fermentation process to maintain nutrient sufficiency, and the culture is harvested at the end of the batch cycle. Optimisation of feeding strategies has been a major factor uting to improvements in growth and productivity in recent years.

Steps in a fed-batch process

Cell culture is typically done in stirred stainless steel tanks. Airlift reactors up to 5000 L scale have also been used but are much less common [39]. Detailed aspects of the issues taken into account in reactor design are summarised by Varley and Birch [40]. It is normal to control pH, dissolved oxygen concentration and temperature in reactors and careful consideration has to be given to the design of vessels to ensure adequate mixing and mass transfer of oxygen and CO2. By way of example, addition of pH control agents to the surface of the culture can lead to high local departures from the setpoint pH value [41]. This can be avoided by adding the agent in a well mixed zone of the reactor. It is important to have laboratory and pilot reactors that can be used to predict large-scale reactor performance. Dissolved oxygen concentration is regulated by the controlled sparging of air into the fermenter and pH is controlled by the addition of CO2 gas or an alkaline solution (e.g., NaOH) as relevant.

Culture media

There has been rapid progress in recent years in the development of serum-free media, driven principally by concerns about the possible introduction of adven- titious agents in animal derived ingredients. Initially, serum was replaced by serum proteins such as albumin, transferrin and insulin, and latterly it has been possible, in many cases, to develop chemically defined, protein- free media which contain no animal derived materials. Frequently, the serum protein was acting as a carrier of an essential nutrient such as a lipid, which could be used to substitute for the protein. Defined media are inherently less expensive and make downstream processing more straightforward as there are fewer contaminants to monitor and remove. Process optimi- sation is also easier in a defined environment. Protein- free culture media for the commonly used industrial cell lines such as CHO and NS0 are commercially available and there are descriptions of such media in the literature (e.g., [33, 42, 43]). For suspension culture, it is common to add a synthetic polymer to the culture medium to act as a protectant against the damage to cells that would otherwise be caused by sparging gases into the reactor [44]. Antifoam is used to prevent foaming caused by sparging.

Table

The evolution of a fed-batch process for antibody producing GS-CHO cell lines showing the improvement in productivity

Process	Antibody (mg/L)	Antibody (fold increase)	Volumetric productivity (mg/L/day)
Original cell line (CHO-K1), original process	139		13
Iteration 1	334	2	33
Iteration 2	585	4	42
New cell line (CHOK1SV), iteration 2	1917	14	128
Iteration 3	2829	20	189
Iteration 4	3560	26	237
Iteration 5	4301	31	215
New clone, iteration 5	5520	40	240

Optimisation of culture conditions

Improvements in recombinant protein production in cell culture have been dramatic. In a review of progress in this area, Wurm [3] noted that concentrations have increased 100-fold since 1980. Some of this progress has resulted from improvements in expression technol- ogy and strain selection as noted earlier, but an equally significant contribution has come from optimisation of the fermentation processes. In Fig. 4, we show current growth and productivity profiles for a GS-CHO cell line making a chimeric antibody compared with data for a process we were using in 1990. Productivity is over 100-fold higher in the current process. It can be seen that there has been a dramatic increase in cell concentration in the reactor and the cells can be maintained in a viable state for a much longer period. This is crucial because productivity is a function of the specific production rate of the cells and the integral of the viable cell count with respect to time (IVC). Cell concentrations exceeding 107/mL are now commonly seen in reactors.

Much of this improvement has come from the optimisation of media and feeds based on a better quantitative understanding of cellsT nutritional requirements. This understanding is often based on iterative analysis of nutrient depletion followed by supplementation of the relevant nutrient in the medium or feed. Strategies for medium and feed development have been reviewed [5, 45]. The overall aim of the fed-batch approach is to increase maximum cell concentrations in the reactor and to prolong the production phase. Dempsey et al. [46] and Sauer al. [47] give examples of NS0 and Sp2/0 processes in which they improved productivity by approximately an order of magnitude.

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