Bioprocesses for High-Density Cell Cultivation in Suspension
Optimized recombinant cell lines derived from CHO and NS0 are now grown in single-cell suspension cultures to densities of
10 x 106 cells/mL or higher during batch and extended-batch processes, a five-fold increase in cell density compared to the production
processes of the 1980s. Some of these parental CHO and NS0 cells may have been genetically engineered to facilitate growth
to high cell densities, but this is surely not a requirement, because such densities can be obtained in naïve cell lines,
as has been observed in the authors' laboratory and by other groups.2 As an alternative to genetic host cell engineering, the parental cell line or its recombinant derivatives typically are
selected from many different phenotypes of cells obtained when identifying cell lines that show characteristics applicable
in bioreactor scale-up scenarios and growth under high-density situations during the production phase.
Improvements in Media Formulation
High-density cell cultivation in suspension has also been made possible through improvements in media formulation. The media
used for today's bioprocesses frequently are chemically defined and lack animal-derived components. Most protein manufacturers
use in-house formulations rather than commercially available ones. Unfortunately, medium development has to be done on an
individual basis for each recombinant cell line. This is a time-consuming task as the effects of multiple components must
be tested alone and in various combinations. For this reason, multifactorial design has been implemented to streamline the
optimization process. In addition, different formulations are necessary for different phases of a manufacturing process, each
one designed for a specific phase of the process. Typically, batch and extended-batch processes can be divided into two phases,
one for rapid growth to a high cell density and the other for protein production under conditions in which the cells are maintained
with little or no growth. Therefore, the dilution and feeding strategies for culture scale-up and maintenance must be carefully
planned, based on an understanding of the growth and metabolism of each individual recombinant cell line. The improvements
in media formulation and in bioprocess control undoubtedly have contributed extensively to the prolongation of the viability
of high-density cultures, but how different manufacturers have optimized their large-scale production processes can only be
guessed. Furthermore, the maintenance of cells at a high density without cell growth can be achieved by different means including
temperature reduction or addition of chemicals that interfere with cell physiology resulting in viability enhancing and stabilizing
consequences.
Improved Host Cell Viability
Protein production also has been increased through improvements in the robustness of recombinant cell lines so that they retain
viability for long cultivation times. Increasing the resistance of cells to apoptosis probably plays a role in the prolonged
production phases now observed for extended-batch cultures (i.e., up to three weeks). How increased resistance to apoptosis
is achieved may differ from one manufacturer to another. There have been many published accounts of enhanced cell survival
and productivity as a consequence of the overexpression of exogenously provided anti-apoptotic genes such as Bcl-2 or Bcl-xL.7 However, the gains in cell viability and recombinant protein production reported in these studies only have been modest.
Therefore, it is not clear that this strategy has played a significant role in the increased viability of recombinant cell
lines used in manufacturing processes. Alternatively, parental cell lines or recombinant derivatives with elevated resistance
to apoptosis may have been selected, based on survival under cultivation conditions with increased cell stress. This approach
may be more likely to succeed than the host-engineering strategy, given the complexity of apoptotic pathways. Moreover, host
engineering by overexpression of exogenous genes may have unintended consequences with regard to other cellular functions,
resulting in cells with undesirable phenotypes. The most important impact, in the opinion of the authors, is a well-balanced
match of the cells' needs at specific phases of the process and optimized media and feeds provided throughout an extended-batch
production phase.
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