Subcloning Services
Overview of hybridoma cell line maintenance and stabilization.
Antibody production by hybridoma cell lines is inherently unstable due to variations in chromosomal assortment. Many cell lines are unstable upon creation and are stabilized by cloning in the early stages of development. Lines such as these can have a high reversion rate to non-secretion unless properly maintained. Another factor that can cause cell line instability is length of time frozen in LN2. Cell lines frozen for greater than 5 years may need extensive cloning upon thawing to recover adequate antibody productivity. All hybridoma cell lines must be maintained by periodic subcloning and cell banking to insure that 100% of the population is producing the desired antibody. Failure to maintain cell lines will lead to loss of antibody productivity and eventually the cell line itself.
BPI Subcloning Program
Bioprocessing offers a comprehensive 3 Phase program to insure that cell lines that go through our process will give rise to stable, antibody secreting, subclones.
Subcloning of Hybridomas with screening
Catalog #:9990-0400
Cell lines that have been frozen for more than five years, have a known history of instability or are of undetermined status, should be subcloned prior to being used for production. BPI catalog number 9990-0400 is a process in which the cell line is subcloned twice by limiting dilution.
At the end of this process a determination will be made as to whether the cell line exhibits stable antibody secretion. Subclones are also screened for growth rate and amount of antibody produced. There are many instances where a hybridoma will have a 100% secretion rate and will not need any further work than 2 subclonings. At this point a subclone exhibiting a high growth rate and superior antibody production will be selected for cryopreservation and further cell banking. Cell lines that do not meet the 100% criteria will have a small freezer lot prepared in order to preserve the subcloning work already done. In order to stabilize the cell line and make it production ready, it must be put through a more extensive cloning process.
Additional Stabilization of Hybridomas
Catalog #:9990-0440
Cell lines that still exhibit less than a 100% secretion rate after 2 rounds of cloning will be considered to be unstable and will have to go through a more rigorous cloning process. This process, catalog # 9990-0440, allows for four additional rounds of subcloning using methods to recover productivity from cell lines that may have less than 20% of their cell population producing antibody. Cell lines that come through this process successfully will have a subclone selected for superior growth rate and antibody secretion. The hybridoma line will then be ready for cell banking.
See Phase III for the process to stabilize cell lines that are still found to be unstable after Phases I and II are completed. Bioprocessing will evaluate the cell line and give a recommendation as to whether or not the cell line is a candidate for phase three as there may be a possibility that phase 3 will not be successful in fixing the cell line stability issues.
Refusion of Hybridomas
Catalog #:9990-0480
A cell line that still exhibits extreme instability after 6 rounds of subcloning can be stabilized by re-fusing the cell linewith a myeloma and allow the chromosomes to re-assort from the beginning. Extensive subcloning is required to selectstable hybrids. Antibody functionality testing is also included in this process. BPI will work with the customer to determine the best method to perform functionality testing.