Elsevier

Journal of Biotechnology

Volume 130, Issue 3, 30 June 2007, Pages 282-290
Journal of Biotechnology

The role of p21cip1 in adaptation of CHO cells to suspension and protein-free culture

https://doi.org/10.1016/j.jbiotec.2007.04.012Get rights and content

Abstract

The up-regulation of cyclin-dependent kinase inhibitor p21 has been shown to enhance productivity of monoclonal antibodies and has been linked to various regulatory processes. To identify the potential role of p21 in adaptation to suspension and protein-free cultures, we studied the survival and growth of anchorage- and serum-dependent CHO cell lines that differed only in the period of p21-induced arrest. p21 overexpression led to rapid adaptation of cells to suspension and protein-free cultures. The period taken to achieve adaptation was correlated with the time the cells were arrested after transfer from the monolayer and serum-fed culture. Interestingly, cell aggregation associated with protein-free suspension culture was reduced in p21 culture in response to the loss of cellular adherence. The processes of adaptation to suspension and arrest did not decrease monoclonal antibody productivity. In contrast, following adaptation to protein-free growth media, an overall increase in specific productivity was observed. The ability of cells to survive in protein-free suspension cultures was due to the requirement of G1 cells to growth factors and to their relatively high resistance to the hydrodynamic forces. This improved process has the advantage of reducing the duration of critical path activity for developing CHO commercial cell lines from 72 to 36 days.

Introduction

Animal cell cultures have been widely used to produce therapeutic protein products due to the compatibility of posttranslational product modification with the therapeutic target; however, the need for increased yields and the requirement to eliminate animal-derived elements are driving industry forward in the search for suitable methodologies that can sustain high-yielding cell lines and speed up the development and production process.

Anchorage-dependent cells must be adapted to grow in suspension cultures due to the scale-up limitation associated with monolayer cultures. Cells which grow in suspension are readily scaled up to production level bioreactors, which are designed to reduce hydrodynamic forces and heterogeneity (Al-Rubeai et al., 1995, Ludwig and Andreas, 1992, Keane and Julian, 2003). Anchorage-dependent cells can be made to be sufficiently robust in the bioreactor suspension environment by a time-consuming adaptation process that includes the addition of protective agents, such as Pluoronic F68 to the media (Kilburn and Webb, 1968, Al-Rubeai et al., 1992). While the effects in suspension culture of hydrodynamic forces and the sensitivity of mammalian cells to hydrodynamic stress have been fully examined, and while the mechanism is well understood (see for example, Hu and Wang, 1986, Peterson et al., 1988, Handa-Corrigan et al., 1989, Oh et al., 1989, Croughan et al., 1989, Jordan et al., 1994, Kunas and Papoutsakis, 1990a, Kunas and Papoutsakis, 1990b, van der Pol et al., 1992), the adaptation process has remained essentially unchanged.

Interestingly, it would appear that the susceptibility of any given cell to the forces exerted by the bioreactor environment are increased by, for example, decreased serum concentration (Kunas and Papoutsakis, 1990a, Kunas and Papoutsakis, 1990b, McQueen and Bailey, 1989), culture medium viscosity (Croughan et al., 1989), and increased cell size (Al-Rubeai et al., 1995). The role of cell size in determining cellular growth, productivity and response to the bioreactor environment may have not been fully appreciated, but cell size regulation is shared with the cell cycle, which, in effect, determines the metabolic potential of a cell and, indeed, its response to the environment.

The ever-increasing need for improved biological safety is pushing industries forward in the development of new serum- and protein-free media formulations. Cell lines will have to be adapted to grow and produce the required product in a defined chemical environment. There have been several approaches for the adaptation of cell lines to the required environments (Kovar and Franek, 1984, Radford et al., 1991, Keen and Steward, 1995, Sinacore et al., 1999, Ozturk et al., 2003) either by a gradual weaning of cultures via the reduction of serum over a set time period or by direct withdrawal of serum. In both cases, the process of adaptation can be extremely long and costly, depending on the cell line and medium selected. Therefore, a process that can speed up the rate of adaptation would provide a valuable tool for both industrial and research applications.

Overall control of the cell cycle, and thus cellular proliferation, is based on the activity of cyclin-dependent kinases (cdks), which are activated by their association with cyclin regulatory subunits via phosphorylation and may, in turn, be inhibited by the alternate binding of cdk inhibitors (ciks), such as p21. The effect of p21 activity in terms of its effect on productivity in cell culture is well documented (Fussenegger et al., 1998, Watanabe et al., 2002, Bi et al., 2004, Ibara et al., 2003); however, it is the ability of p21 to induce arrest that is of particular interest in this study, which examines new roles for p21 in process development.

Examination of the proteins and pathways involved in responding to the stresses caused by chemical or physical means has shown that an inbuilt adaptive response exists which is capable of adjusting the levels and/or activity of the genome-protecting machinery, usually through the synchronization of cell cycle arrest, DNA repair and apoptosis (Hofseth, 2004). By initiating p21 overexpression, cells accumulate in the G1 phase of the cell cycle. This arrest of cell cycle progression may allow additional time for repair of environmentally derived damage before the cell is replicated (Weinert and Hartwell, 1988). We hypothesize that an extended period of cell cycle arrest allows cells to survive in a phase where extracellular growth factors are not needed, and/or the accumulation of extracellular autocrine growth factors to a level that can support proliferation. It may also allow the resulting genome damage, created through the direct withdrawal of serum, to be repaired prior to cell cycle release. Interestingly, recent studies also indicate that p21 expression may also provide some protection against apoptosis during the process of adaptation to genotoxic stresses. A recent study (Roninson, 2002) showed that wild-type p21 has the ability to inhibit apoptosis and stimulate transcription of secreted factors with mitogenic and antiapoptotic activities. In this paper, we examine the effect of p21-induced cell cycle arrest on the ability of an anchorage-dependent CHO cell line to survive and adapt to a suspension and protein-free growth environment whilst maintaining productivity.

Section snippets

Cell-line maintenance

The recombinant Chinese hamster ovary cell line CHO3B2 was previously created by transfecting the mouse–human chimeric B72.3 IgG4 antibody expressing cell line 22H11, previously obtained from Lonza Biologics, with an inducible copy of p21cip using the LacSwitch system (Bebbington et al., 1992, Bi et al., 2004).

Cells were maintained for several passages prior to experimentation in DMEM F12 minus glutamine supplemented with 10% heat-inactivated dialysed fetal calf serum (dFCS) (PAA, Yeovil, UK),

The effect of p21 overexpression on CHO cell-line adaptation to suspension culture

In order to establish whether a period of cell cycle arrest would have an impact on the ability of industrially relevant CHO cell lines to adapt to single-cell suspensions in serum-supplemented growth environments, four cultures of the transfected p21cip1-expressing cell line were set up as described in Section 2. The results obtained (Fig. 1) clearly demonstrate the impact of 3-, 6- or 9-day cell cycle arrest in comparison to the nonarrested control culture. Cultures that were maintained in a

Discussion

In this study, we have demonstrated that p21cip1 overexpression can significantly reduce the time necessary for the adaptation of an anchorage-dependent CHO cell line to suspension and protein-free growth environments without compromising the growth or productivity of the cell line.

A previous study (Al-Rubeai et al., 1995) has clearly demonstrated that cells in the G2/M phase of the cell cycle are more susceptible to mechanical damage than cells in the G1 phase. The authors have concluded that

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