The development and manufacture of recombinant therapeutics requires the production of stable, high-yielding cell lines. The success of this process depends to a large degree on both the method chosen for transfection and clone selection, and also the cell line selected for culture.
The identification of high-yielding cells has traditionally been a lengthy process that involves sorting through thousands of clones in search of the few that, to some extent by chance, express the recombinant protein at suitably high levels. While optimizing media, feed strategy, and bioreactor parameters play an important role in ensuring maximum protein production, the optimization process starts at the very beginning, at the level of DNA.
The site of gene integration during transformation can determine the levels of protein produced—if a gene integrates into an area of the host genome that is transcriptionally silenced (heterochromatin), expression levels will be low. Genes that are constantly or regularly active are found within euchromatin and have high levels of histone acetylation. In contrast, heterochromatin is generally characterized by extensive histone deacetylation and high levels of histone methylation.
A number of strategies have been developed to change the structure of chromatin surrounding the integrated gene in order to circumvent the issue of integration site-specific repression.
In collaboration with King’s College London, Cobra Biomanufacturing has identified short polynucleotide sequences encompassing genetic regions surrounding housekeeping genes that maintain an open chromatin structure, thus allowing high levels of transcription of the subsequent genetic sequence. The DNA elements have been termed ubiquitous chromatin opening elements (UCOEs) and now are proving to be of great value in bioprocessing.
UCOEs consist of methylation-free CpG islands that encompass the promoters of housekeeping genes, for example the dual divergently transcribed heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1)-heterochromatin protein 1Hs-γ (chromobox homolog 3, CBX3) promoters. CpG island-containing fragments of 4–8 kb from the HNRPA2B1-CBX3 (RNP) loci have been incorporated into eukaryotic expression vectors and have resulted in a 10- to 25-fold increase in productivity compared to vectors without a UCOE insertion.
The UCOE is always positioned upstream of the typical expression cassette, consisting of a cytomegalovirus (CMV) promoter, the gene of interest, and a polyadenylation sequence. Transfecting cells with such a vector results in a vastly increased proportion of high expressing, stable clones in the cell population, making the process of clone selection and culture much faster and easier. For antibody production, a dual UCOE vector is used with separate cassettes for heavy and light chain expression. Following transfection, the linearized UCOE expression cassette typically integrates into the chromosome at 5–10 copies per cell.
The value of this technology to the bioprocess industry is, however, dependent on more than just the successful transformation and isolation of clones—cell line selection and development are equally important.
Cobra Biomanufacturing has developed the maxXpress system, which combines UCOE-containing clone production with custom cell-line development. The cell line used should be suited to the specific production task proposed. Requirements include a cell line that rapidly grows to high cell numbers in a suspension setting, in chemically defined or protein-free media. The cell line would also need to be easy to transfect and be susceptible to standard selection protocols. It would need to be easy to subclone and also express the recombinant protein at high levels with the desired secondary modifications.