HOST CELL ENGINEERING
A number of reports are available that demonstrate the use of genetic engineering to improve the growth and product formation
potential of the host cell. The integration of several anti-apoptotic genes including E1B-19K and Aven (in CHOcells) have
been reported to improve the performance of mammalian cell culture.21 The other examples include the over expression of cell cycle regulatory proteins cyclin E and E2F-1 independently to activate
the proliferation of CHO cells in the absence of serum and external growth factors.22 The co-expression of cell proliferation genes and anti-apoptotic genes has also been reported to improve cell culture performance.23 Targeted approaches to affect the cell adhesion,24 antibody secretion,25 and post-translational modifications of antibodies26 have also been demonstrated.
PRODUCING A HIGH PRODUCER
The production scheme of generating a clonal population to produce the protein of interest is well established. The host cell
is transfected with the plasmids containing the recombinant gene with the necessary regulatory elements and the gene conferring
the selection pressure. The most popular selector genes are dihydrofolate reductase (DHFR) and glutamine synthetase (GS).
For these systems, the expression of recombinant proteins can be significantly increased by exposing cells to gradual increases
in concentrations of methotrexate (MTX) or methionine sulfoximine (MSX). The surviving cells will frequently contain significantly
higher copies of the integrated plasmids in host chromosomes and will produce more protein compared to unamplified cells as
a result. However, the specific productivity will vary among clones and the identification of high producers will require
the screening of hundreds or thousands of cell lines.
Table 1 depicts the evolution of the productivity of mammalian cell lines over the last 20 years as a result of cell engineering,
media optimization, and bioreactor process development efforts.
Table 1. Productivity increases over the last 20 years in mammalian cell culture
SINGLE CELL CLONING
The survivor cells after selection are often subjected to serial dilution in secondary containers like 96-well plates, and
the resulting cells that show as single colonies subsequently are expanded in larger containers like 12-or 24-well plates.
The selection of the final clone often takes a long time because the clonal stability is as important as the protein/antibody
titer achieved with a given clone. The other traits of interest may be the cell sensitivity to osmolality and pCO2, as the cells may have to counteract high osmotic pressure and elevated dissolved carbon dioxide levels at large scale. To
speed up the process of finding the right clone, a number of biotechnology companies have invested in highly automated, commercially
available systems. These systems combine image processing and highly mechanized robotics to identify and pick the mammalian
colonies of interest. The other technique that has gained popularity for single-cell cloning is the cell sorting based on
florescence activated cell sorting (FACS). After the single-cell clones have been isolated, the identification of high producers
still remains a time consuming process. The feasibility of using FACS in conjunction with the use of a nonfluorescent reporter
protein molecule that can be detected by a fluorescent antibody also has been demonstrated to screen the high producers from
the sorted single-cell clones in 96-well plates.27 Although the subject of single-cell cloning has generated a lot of academic interest, the fundamental mechanisms that dictate
clonal productivity and stability are still unknown.