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European Biopharmaceutical Review

Path to Progress

EBR: What do you think are desirable attributes for a recombinant cell line identified during cell line development?

Alison Porter: During cell line development, we wish to identify those lines which show desirable attributes for use in biomanufacturing. These include stable and high product titre, and growth properties which are suitable for the manufacturing process. Another important consideration is the manufacture of product with stable and suitable characteristics. This is especially pertinent for biosimilars and certain non-monoclonal antibodies where information on product characteristics is increasingly being used to drive decisions early on during cell line development.

Describe the key components in a cell line development strategy.

When designing a cell line development strategy, the aim is to identify cell lines with higher titres within shortened development times and in a manner that meets regulatory acceptance. An important prerequisite to an optimised cell line development process is the use of a superior expression system comprising a host cell line and expression vector.

Other key components include: the introduction of platform screening procedures which are relevant to platform production processes; having the ability to demonstrate an acceptable level of monoclonality with a minimal number of formal cloning rounds; creating high expressing cell lines without the need for multiple rounds of gene amplification; and using a process free of animal-derived components.

What would you say are important points to consider when developing a new host cell line?

It is important to select a fully traceable cell line that is both well-characterised and compatible with production requirements so that you can save a substantial amount of development time in the future. Key elements for a host cell line include the ability to grow well in suspension conditions in chemicallydefined media, and the capability for high expression of recombinant proteins. It is also advantageous to have created a fully-characterised and documented current Good Manufacturing Practice cell bank for the host cell line.

During development of a new host cell line, you can end up with a very large panel of potential candidates. It is therefore key to have a well-designed hierarchical screening strategy to gradually decrease these numbers. Any screens used in this process should assess the multiple desirable attributes you wish to identify in recombinant cell lines. This is highly important as a cell line with a particular desirable attribute (for example, high specific growth rate) may not perform so well in other attribute categories (such as low specific production rate).

Explain what the directed evolution approach to developing a new host cell line involves.

Chinese hamster ovary (CHO) cell populations are functionally heterogenous – that is, a mixed population of individual cells, each with its own particular set of functional attributes rendering it either a suitable or unsuitable production vehicle. Indeed, this clonal variation is already exploited during cell line development when large numbers of potential cell lines are screened to isolate those with the most desirable attributes for biomanufacturing. ‘Directed evolution’ based approaches therefore aim to control/manage this clonal variation by either improving the functional capability of, or removing unsuitable variants from, the parental host population.

The selection of CHO cell variants with improved characteristics is typically an iterative process comprising several rounds of induced selective pressure. Examples include relatively simple approaches, such as extended cultivation of cell lines in altered subculture regimes or limiting dilution cloning. Certainly, efforts to adapt cell lines to serum-free, chemically-defined media will be a familiar directed evolution approach to many. Other more complex examples include using fluorescence-activated cell sorting to enrich for cells with extended viability and growing cells in chemostat culture.

How does this vary from other methods, and what are the benefits of using this approach?

The other obvious path to take when developing a new host cell line is cell line engineering. Directed cell engineering has held much promise for the ability to create functional attributes, albeit in a piecemeal manner. However, the identification of suitable engineering targets has proven difficult, success has been varied, and it is highly likely that generation of an optimal cell line would require a complete redesign. Furthermore, the intellectual property situation around engineering targets and the methods by which they are developed can make cell engineering an undesirable proposition.

In contrast, directed evolution strategies allow us to select cell lines based on their functional phenotype (likely a consequence of the up/down-regulation of numerous genes/pathways) and potentially see the benefit of multiple rounds of directed cell engineering within a shorter timeframe.

What technologies can you incorporate to facilitate selection of the ‘best’ recombinant cell lines?

Efforts to improve the selection of the ‘best’ recombinant cell lines have focused on the introduction of screens early in cell line development that are more comparable to, and therefore more predictive of, performance during manufacturing. Examples include the use of microbioreactors and shaken multi-well plate platforms which are operated in suspension mode and have feeds applied. As product attribute information is also being used to drive decisions during early cell line development, analytical platforms are being introduced with the ability to generate data from a large number of small volume samples in a short timeframe. Both the upstream and analytical technologies are typically linked to automation-based platforms which significantly advance throughput.

However, beyond improving the predictability of the screens performed during cell line development, it is also important to optimise both the host cell line and expression vector. These are often overlooked key production variables which can have a significantly positive effect on the identification of production process compatible cell lines.

How quickly can material be generated during a cell line development programme?

It can take a number of months to establish a stable, clonal production cell line. However, to minimise preclinical development time, there is a desire to produce milligrams to grams of representative product in a shorter time period. Transient expression has been a commonly used technology to generate material for these purposes, but can be low yielding and operationally challenging at large scale.

An alternative strategy is to use stable transfectant pools, which can generate appropriate amounts of product within a couple of months. A stable transfectant pool is a heterogenous population of transfected host cells that have integrated recombinant DNA. They are typically higher yielding (greater than 1g/L) than transient systems, and large-scale production can be achieved by expansion of the culture volume.

A further advantage is that creation of these stable transfectant pools is often the first step when developing clonal production cell lines. Therefore, cryopreserved stocks of transfectant pools, used for supplying material, can be revived at a later date and used as a starting point for clonal cell line generation, thereby shortening the timeline for this stage of development.

Alternatively, clonal cell line generation and production of material from transfectant pools can simply occur in parallel.

How are improvements in media and automation benefiting cell line production?

When looking to introduce screens predictive of manufacturing performance during early cell line development, it is beneficial to use production medium and feeds. That way, cell lines that have demonstrated favourable characteristics in the production medium and feeds at this early stage are immediately selected, and should perform well in the manufacturing process. Using this approach also offers the potential to reduce the need for further medium development in the future. Automation allows larger numbers of cell lines and samples to be assessed in such highly predictive screens.

In which areas should research into cell line development focus?

In the coming years we will no doubt see further demand for higher titres and quicker development times. To meet these goals, we will have to improve the screens we perform during cell line development and introduce further developments in cell line and vector engineering. The main factor preventing advancements in these areas is the limited understanding of how to successfully predict cell line manufacturability during early cell line development. Such an understanding is likely to necessitate the measurement of a number of variables at low sample volumes and further advancements in how we integrate and understand large (perhaps -omic) datasets.

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Alison Porter is Head of Mammalian Cell Culture R&D at FUJIFILM Diosynth Biotechnologies. She recently led the development of the company’s new Apollo™ mammalian expression platform and has considerable industrial experience in mammalian cell culture, specialising in the construction of recombinant cell lines. Previously, Alison spent two years at Bio Products Laboratory, followed by 13 years in the Cell Culture Process Development group at Lonza. She has a PhD in Biotechnology from the University of Manchester.
Alison Porter
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