From the selection of expression technology to clinical production, the correct balance between time and cost versus yield and quality is essential to maximize return on investment. Although creation of a regulatory-compliant cell line is
essential for the production of clinical material, the initial choice of expression technology is often a rushed or overlooked
activity that is overshadowed by financial and/or time constraints.
During recent years, there has been increasing pressure within the biopharmaceutical industry to produce high-quality products
within shorter time lines, which can be achieved using robust, low-cost processes. Many recombinant proteins in the development
pipeline are monoclonal antibodies, which typically require multiple doses at 10–100 times greater dosage than successful
recombinant protein drugs, such as erythropoietin and human growth hormone,1 raising concerns regarding current cell culture production capacity.2
Figure 1
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This limited capacity has resulted in a drive to increase expression levels to >1 g/L, with some now reporting >5 g/L.2 Because of the demand for greater productivity, cell line development and optimization has become a critical step and, to
be effective, should be considered early in development. The parameters for optimizing a cell line may include deciding on
the appropriate expression (enabling) technologies, media choice or development, clone analysis and process optimization.
As with any biopharmaceutical process, the potential benefits of utilizing proprietary expression technologies need to be
carefully weighed against the costs, in particular intellectual property (IP) right encumbrance, which may lead to downstream
royalty stacking.
When producing a clonal cell line, the anticipated development route of molecular biology, transfection/selection and the
minimum of two rounds of cloning accompanied by some scaled evaluation of each clone is often challenged by time lines, cost
and the cell line used. Once a suitable cell line has been established, further development and optimization of the production
process can be undertaken. Different modes of cultivation and the variety of vessels available, including traditional steam-in-place
bioreactors and, more recently, disposable systems, provide further decision points and areas for optimization. Demonstration
of the scalability of a chosen process prior to cGMP production is essential for a rapid and cost-effective route to clinical
material. Addressing the balance of all of these factors as early as possible will eliminate hurdles at later stages, thereby
reaching the desired endpoint more quickly and cost effectively. Expression technologies
On the go...
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This should be considered the most important decision in the development cycle of any product as an uninformed decision based
on commercial requirements alone may, ultimately, have disastrous consequences on the efficacy of the target recombinants.
Bacterial systems are ideal because they are low cost, highly productive and rapid to use compared with mammalian systems.
The absence of adventitious agents, such as viruses, that may be present in mammalian systems is also a regulatory and safety
benefit. In many cases, however, bacterial production cannot yield an effective product because of the requirement for post-translational
modifications, e.g. glycosylation, which are only provided by eukaryotic systems.
Yeast and insect cell production systems combine some of the benefits of microbial production with the capability for limited
post-translational modifications, but some of the best production systems, such as Pichia pastoris, are proprietary and require licences to use.
Currently, an estimated 50% of the hundreds of biopharmaceuticals in the pipeline are developed using mammalian systems.2 The principle advantage is the production of a recombinant protein that is truly 'human-like' in nature, but the production
costs and challenges are greater than for microbial systems.
Table 1 Common expression mammalian systems.
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Another important consideration in an expression system is the choice of vectors. Table 1 details some common mammalian expression
systems that can increase the level of highly productive stable clones. Ensuring licence-free vectors from the beginning of
development avoids royalty stacking and a recloning exercise later in development, which risks changes to product yield and
quality.
Transfection
There are many methods for delivery of expression constructs to cell lines. Efficient delivery of plasmid DNA is variable
across techniques, and consideration for the delivery method should be based on the requirement for transient, episomal or
stable maintenance or integration of the expression construct.
Electroporation is a valuable and effective alternative to other physical and chemical methods. Advantages include:
- It can be used on a variety of cell lines, including prokaryotes, yeast and eukaryotes.
- A higher transfection efficiency is generally obtained compared with the use of chemical reagents.3
- Smaller quantities of DNA are required.4
- The method incurs lower costs compared with expensive chemical reagents.
- No expiry dates are involved.
Clone selection
There will always be demand for cell development services as the process is vital to the end product. However, selecting the
final clone can be complicated and difficult because of the minimal environment in which single cells generated through limited
dilution or facilitated cloning exist. The process is lengthy primarily because of many cell lines having a transfection efficiency
of <1%.5 Most cells need to communicate with surrounding cells to enable growth, but the probability of a single cell multiplying
successfully can be increased by supplementing the media with either serum or preconditioned media.5 The serum can easily be depleted while scaling up, but some cell clones will be lost as they cannot survive without the
serum. The traditional method is to manually perform limited dilution cloning from a transfected pool. This is labour intensive
as it involves initial analysis and consequent monitoring for single cell wells. Typical time lines for each round of cloning
from the point of single cell seeding to evaluation of clone productivity can be 6–12 weeks depending on the cell line and
the recombinant produced. Regulatory guidelines recommend at least two rounds of dilution cloning, and the effort required
for maintenance and analysis is proportional to the number of clones selected from each round.
Many automated systems have been developed to aid cell line development because of high demand and competition. These systems
assess cell growth from a single clone and isolate higher expressers for further expansion, and some can perform media optimization
in parallel. Using an automated system allows a higher number of clones to be analysed and removes any error associated with
multiple operators. Two such systems are the ClonePix (Genetix, UK)6 and Cell Xpress (SAFC Biosciences, UK),7 both of which use fluorescence technology to detect high-expressing cells from thousands. These high-throughput screening
methods can reduce the time taken to create a robust cell line from 12–18 months to 6–12 months, and provide high-throughput
analysis while maintaining and selecting clones.5 Disadvantages include the expense and the lack of a track record, although it is envisaged that these will be overcome with
the increased uptake of technologies.
