In addition to the work done with well-established cell lines, a designer cell program was incorporated to develop new cell
lines of human animal or avian origin from scratch, using different tissues of the respective species. ProBioGen is confident
that new cell lines based on rational design will be able to outcompete traditional cell lines and primary cells in the production
of recombinant proteins and virus vaccine strains. Benchmark results of our cell engineering effort are shown in Table 3.
Table 3. Proven Strategies for Development of High-Yielding Cell Lines
Methods in Brief for the Three Referred Cell Development Programs
(1) CHO DHFR-cells from a qualified source were electroporated with the ProBioGen high-level expression vector containing
the transgene (a human therapeutic glycoprotein) under the control of a proprietary hybrid promoter and flanked by two independently
expressed selection markers. Clone pools were derived employing a dual-selection strategy. Highest producers were established
in a small-scale screening (30 clones). The complete process — from selection of highly productive cell pools, to cloning
of single cells, to selection of a high producer cell line — was performed in ADCF basic medium with GMP-compliant protein
supplements. The secreted protein was measured in a specific sandwich ELISA and calculated on a per cell and day basis.
(2) The human mouse heteromyeloma CB03, secreting large amounts of IgM, was transfected by electroporation with a targeting
vector containing the leptin Fc-fusion protein gene. The vector was designed to allow for homologous recombination within
the Igµ locus. Through targeting, the endogenous Ig promoter was replaced with a CMV-EF1alpha promoter. Homo-logous recombinants
were detected by the absence of Igµ expression and the presence of the fusion protein, verified by PCR. After recloning and
identifying a stable, high-producer, the secreted fusion protein was measured in a specific sandwich ELISA test in a stationary
(3) A human neuronal designer cell line, derived from primary cells in a fully documented process, was transfected with Effectene
(Qiagen, Germany) for stable expression of AAT from a proprietary hybrid promoter. A standard selection marker (puromycin)
in an independent transcription unit was used to screen for a small number of high-producer clones. The product, which secreted
over 72 h from stationary culture in T flasks, was quantified by a specific sandwich ELISA.
Once 40 pg/c-d can be reached for a certain protein in standard cell culture in CHO, media selection and process optimization
is expected to increase volumetric productivities in excess of optimum-performing, fed-batch processes. The future potential
of designer cell lines derived from scratch is illustrated by the results obtained so far. A comprehensive summary of current
experiences of the cell-development team is described in detail elsewhere.6
PILOT-PLANT CAPACITIES AT A GLANCE
Figure 6 illustrates the boosting of plant capacities from 650 g of crude product (without purification) for low-expression
cell lines to more than 5 kg crude protein per year. We assumed the use of cell lines with cell specific productivities of
40 pg/c-d for batch manufacturing.
Figure 6. Comparison of Annual Plant Capacities Extrapolated for Low-and High-Yielding Cell Lines