Biology Reference
In-Depth Information
Figure 2. An example of single-cell cloning technique for mammalian cells.
The cell bank is the primary source for the recombinant protein produced,
so verification that the correct molecule is being produced must be done at the
inception of the bank. Confirmation of the DNA and/or RNA coding sequence
ensures that the cells encode the required genetic sequence for the protein.
Verification of the genetic sequence within the cell must be followed with ver-
ification of the amino acid sequence of the purified protein. Additionally,the
cell bank must be examined to confirm that it is not producing any altered
forms of the protein. For example, during gene integration in mammalian cells
(after transfection or amplification), the cell's genome is rearranged,and
rearrangement possibly may involve the coding sequence of the protein of
interest. One concern is that the rearrangement could occur at a site that results
in a molecule containing a portion of the correct protein and a portion of anoth-
er protein. In this event, the rearranged molecule could retain some of the cor-
rect protein's characteristics and be carried through a purification process;
however, the net result may be either the incorrect protein or a mixture of cor-
rect and incorrect proteins. Therefore, the cell bank is examined in great detail
to ensure the absence of rearrangements that could lead to unwanted proteins
being carried through manufacturing.
Ensuring that adventitious agents are not present is crucial. When a cell
bank comprises mammalian cells, which theoretically can act as a host to
viruses and is the starting material for every lot of protein, the cell bank is
examined for mycoplasma, bacteria, fungi, and viruses. Numerous
in vitro
and
in vivo
assays are used to assess viral contamination. In the end,all of the
aforementioned tests for adventitious agent must be negative. If they are not,
the cell bank cannot used to make material intended for clinical use.
