PANGEN¡¯S PROPRIETARY CHO CELL EXPRESSION SYSTEMS

High Speed: Shortened screening process

   A few eukaryotic proteins have been expressed efficiently and inexpensively in prokaryotic hosts.  However, many eukaryotic proteins synthesized in bacteria exhibit low specific activities due to the incorrect folding and post-translational modifications, mainly glycosylations.  Because of these problems, considerable effort has been made to develop mammalian cell expression systems such as COS cell system, CHO cell system, and viral vector system, to express mammalian proteins in mammalian cells.  Even though these systems are possible for the production of the recombinant proteins, CHO system has been frequently used up to the present to produce a large amount of protein for a long period of time.  However, the main difficulties in CHO system lie in the laborious selection processes due to the poor frequency of desired stable transfectants, and a low expression level of target genes during the cell cultivation.  Moreover, for the overproduction of foreign proteins from CHO cell, the methods of gene amplification for the transgene has been used to maximize the desired expression levels, such as using DHFR(-/-) CHO cell with the gradual increase of the selective drugs.  In contrast, PanGen's new system results in high yielding cell lines after only two steps of MTX selection, which greatly shortens the screening time.

   In the conventional CHO cell technology multiple colonies have to be separately cultured in a prolong process to obtain the best one, since it is often difficult to know which colony is the best until all the colonies are fully grown.  In most cases using PanGen's vector, significant amounts of desired protein can be synthesized from the pool of transfectants prior to colony isolation.  About 8 to10-fold increase in protein production is observed during the early stage.  A large proportion of transfectants (70-80%) synthesize the recombinant protein at a relatively uniform level as the expression seems to occur in a position-independent and copy number-dependent manner.

   Consequently, the laborious manipulation of cell culture could be minimized.  In addition, Semi-automation of the whole cell culture process is currently underway, which will reduce the labor cost even further.  Stable CHO cell lines for biopharmaceutical productions can be obtained in 4-6 months.  Proteins suitable for various research purposes can be obtained in sufficient amounts in usually 2-3 months.

High Expression Level

   Conventionally the production level of a protein from the animal cells is often very low and sometimes unpredictable.  It may relate to the inefficient activation of integrated gene expression, where the inefficiency is strongly dependent on neighboring sequences context.  The PanGen's expression system is believed to confer position-independent expression of the integrated sequence.  Regardless of the integration site and nature of surrounding sequences, the productivity seems to be primarily dependent on the copy number of amplified gene alone.  The presence of an efficient transcription terminator in PanGen's new vector eliminates problems associated with possible read-through and improves the stability of mRNA, which leads to increased protein yield.

High Stability

   Sometimes a marked decrease in the protein production is observed during long-term culture.  The presence of selective agent such as MTX is essential to maintain the amplification of introduced sequence.  In PanGen's expression system, amplified sequence is stably maintained in the absence of selective agent.  The use of MTX, a potential carcinogen, can be avoided for large-scale production of therapeutic proteins.