The Ciliate vantage for biopharmaceuticals

The Ciliate performance expression system (CIPEX-System):

Feasibility- and evaluation studies demonstrate the broad applicability of the Ciliate Performance Expression System.

The CIPEX-System is an innovative expression platform for highly productive manufacturing of protein based biopharmaceuticals.
CIPEX is based on apthogenic eukaryotic protozoan organism Tetrahymena, which combines in an ideal way the advantages of different conventional expression platforms. The biological safety of the CIPEX-System, its cost-effective and productive fermentation procedures as well as the feature to produce glycosylated proteins present the ideal solution for manufacturing of complex recombinant proteins. Furthermore secretion of the proteins substantially simplifies the purification of the protein product.
Cilian AG was able to prove the functionality of its platform technology CIPEX by the heterologous expression of 14 pharmaceutically relevant proteins within the frame of feasibility and evaluation studies, both with industrial partners or internally.

The studies were performed for secreted as well as membrane-bound or intracellular proteins. The volumetric productivity is at least as good as conventional expression systems.
Beside genetic and analytical methods, different up- and downstream procedures were developed and successfully adopted to meet the requirement of target proteins.
Consequently, the R&D-team of Cilian AG possesses thorough knowledge base in the area of the recombinant protein production with Ciliates. The professional competence of our R&D-team enables the scale up to industrial production.

Process development and the recombinant production of complex proteins of interest using CIPEX like e.g. viral antigens or monoclonal antibodies are offered as a service. For more details please see here. ⊂Service Offer⊃.


Advatages of the CIPEX-Systems:

Efficient production of complex proteins

The CIPEX-System is a eukaryotic expression system whose protein biosynthesis is comparable to higher animal cells. Recombinant proteins become translationally modified and processed. This includes amongst other things the formation of disulfide bonds, GPI anchors and N-glycanes.

The post translational protein modification is an essential requirement for the correct folding and functionality of complex biopharmaceuticals.

Secretion of target proteins

Tetrahymena is able to secrete proteins into its surrounding medium. Purification from culture supernatant is much easier and cheaper than from cells. For protein targeting special targeting sequences from Tetrahymena or from the target proteins can be used.

Consistent glycosylation

Our production organism synthesizes proteins with a highly consistent biantennary oligomannose-N-glycosylation structure. Especially for biopharmaceuticals like therapeutic monoclonal antibodies a consistent glycosylation is a crucial quality feature. It leads to higher stability in the blood stream, minimizes the risk of side effects and will lead more likely to the desired therapy effect.

High productivity

The space-time-yield is one of the pivotal features of a recombinant protein production process. The CIPEX-System reached space-time-yields comparable to established expression systems in several proof of principle studies.
Without any elaborate clone screening Cilian AG reached space-time-yields of 10 mg/l per day for monoclonal antibodies; and 60 mg/l per day for vaccine proteins.

Advantages in process technology

Cultivation of Tetrahymena can be performed with well established fermentation techniques (batch, fed batch and continuous fermentation) to achieve high cell densities (up to 2 x 107 cells/ml). Similar to yeasts our production organism is characterized by short generation times of about 1,5 to 4 hours.

The protein production can be carried out in standard fermentation facilities for microbial expression systems. No expensive adaptation of existing production infrastructure is necessary. Scale up to volumes up to 1000 liters has already been demonstrated.

Tetrahymena features fast growth on inexpensive peptone and chemically defined media without any supplementation of peptide or serum components. The stationary growth phase is reached after approx. 30 hours of cultivation. Media prices for Tetrahymena are expected to be one order of magnitude lower compared to media for animal cell cultures because of the absence of expensive supplements like peptides or serum.

High biological safety

A further aspect of great importance is the biological safety of Tetrahymena. Due to its high AT-content and its unusual codon usage viruses from higher organisms cannot propagate in Tetrahymena. Furthermore Tetrahymena is a well examined model organism. Even after 50 years of basic research no viruses have ever been observed.
Tetrahymena strains were examined in cooperation with independent institutions and no pathogenic agents like viruses or mycoplasms were found in approved indicator cell lines. A proliferation of viruses from higher animals or humans can thereby almost certainly be avoided in a production process using Tetrahymena.

In contrast to industrial processes with human or animal cell cultures, expensive virus clearing is not necessary. This is a major difference between Tetrahymena and mammalian cell lines like CHO, BHK, NSO or human cell lines in the pharmaceutical production which have to be protected utilizing complex and expensive procedures against contamination by endoparasites.

For this reason the biological safety of Tetrahymena as a production organism for therapeutic proteins is superior to animal or human cell cultures.

Established molecular biological tools

Optimized, episomal replicated high copy number vectors or integrative systems are available for the protein expression with the CIPEX system. High copy number vectors are based on rDNA mini chromosomes of Tetrahymena where up to 9,000 copies per cell lead to a high gene dose and an efficient protein biosynthesis. Depending on the promoter, the protein expression can be carried out constitutively or by induction. Furthermore, an efficient protein secretion into the supernatant is possible by the using different protein targeting sequences.

The high AT-content of Tetrahymena is an additional, native advantage on the molecular biological level. Genes from organisms with AT-rich genomes can be expressed by the CIPEX system without the need of synthetic genes (example: Plasmodium and other protozoa with AT-rich genome)