Clone Pool Concept

Cell Lines for Therapeutic Protein Manufacturing

The development of high producer cell lines for the manufacturing of recombinant proteins and monoclonal antibodies requires the identification of rare clones which combine efficient transcription with superior folding, processing and secretion capabilities.

ProBioGen's careful vector design combined with the use of novel promoters, which are resistant to inactivation regardless of their position in the genome, and a sequential double selection strategy, allows the reproducible generation of highly productive clone pools. These clone pools are suitable for the production of test material prior to single cell cloning. The approach also improves the identification of superior clones.

ProBioGen uses starter cells which have been selected for efficient growth in media free of animal components. This provides the basis for production cell clones that achieve superior cell densities after a minimal lag phase.

In addition, ProBioGen scientists further modify these cell lines by metabolic engineering and interference with apoptotic signalling to increase the specific cell productivity which makes the cells ideally suited for industrial cell culture processes.

Clone Pool Concept

What can you do to make your discovery programme more efficient and successful, when instead of one gene candidate for a therapeutic protein, you have to decide among three candidates?

Two strategies are used typically to address this issue:

A) Small amounts of protein from each gene can be produced using transient transfection to enable the performance of partial in vitro tests and subsequent decision on one winner gene to be used for the development of a producer CHO cell line.

B) Three producer cell lines can be developed and used for the production of enough protein material for a well-founded decision.

Whereas the first solution takes more time for the entire process and provides unsatisfactory quantities of proteins for decision making, the second solution is very expensive and thus unrealistic. .

C) A unique third solution could be created using ProBioGen's specific vector composition and selection strategy, which allows the development of clone pools that have high average cell productivity:

Using the DNAs in question, up to three stable high yielding producing clone pools can be developed. These clone pools are then used to produce 300 mg - 3,000 mg of recombinant proteins each. After in vivo and in vitro testing of the proteins, the chosen winner clone pool can be further developed into a full producer cell line.

This new alternative offers the necessary product quantities to allow the generation of enough data for a well-founded decision on the product candidate for continuing development, whilst keeping the costs low and the development time on track.

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Cell Lines for Therapeutic Protein Manufacturing

ProBioGen's cell line development starts from CHO dhfr (-) (both DUXB11 and DG44) or NS0 cell banks that have been generated in the absence of serum.

Fully documented cell lines are developed in a 6-7 month period.

The process starts with the generation of multiple clone pools by sequential double selection.

From these clone pools, single clones are isolated using single cell cloning. Up to 1,000 clones are tested for productivity. Using mini-shaker cultures, a subset of clones is analysed for specific productivity, product integrity and growth properties in GMP production medium.

If desired, glycosilation analysis for individual clones is performed to identify preferred candidates. Homogeneity of expression within the clone is analysed as a predictive parameter for clone stability.

A secondary cell cloning step for 3 primary clones and seed cell banking concludes the development.

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