Results of a recent study sponsored by Bio-Rad (www.bio-rad.com) demonstrated the effectiveness of CHT for viral clearance. More than 1,000-fold clearance of xenotropic murine leukemia virus (X-MuLV) and 100-fold clearance of minute virus of mice (MVM) were realized.
The selection of viruses is in compliance with the recommendations provided by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, as well as in the Committee for Proprietary Medicinal Products documents on virus validation studies. The clearance data is shown in the Table.
These results were achieved with protein-A-purified human IgG1 Mab loaded on a column of CHT type I, 40 microns. The sample was mixed with virus, applied in 10-mM sodium phosphate, pH 6.5, and then eluted with a 10-column-volumes linear gradient to 2-M NaCl. The column was then cleaned with 10 column volumes of 0.5-M NaPO4, pH 6.5. These conditions are consistent with the range recommended for removal of aggregates, leached protein A, DNA, and endotoxins.
The X-MuLV (an enveloped retrovirus) data are most relevant because of the potential for endogenous retroviruses to be present in the CHO cell lines that are used for antibody production and for other mammalian protein production. Thus, these data are of universal interest to purification process developers.
MVM is a nonenveloped parvovirus with high resistance to physicochemical inactivation. It is not associated with mammalian production systems but is used to establish the ability of the process step to remove a virus rather than to achieve a specific removal goal.
Elution of the majority of both viruses in the 0.5-M phosphate cleaning step is very positive because it indicates that a similar degree of virus clearance can be expected from different Mabs, as long as they are eluted within the NaCl gradient. Every Mab examined up to this date can be eluted with a NaCl platform, and the process-development strategy suggested ensures that other Mabs will follow this pattern.
These data also indicated that equivalent viral clearance can be obtained when CHT is conducted in a flow-through mode, as long as the phosphate concentration is kept low and selectivity is controlled with NaCl. The data presented demonstrate the feasibility of using CHT for viral clearance, but it will remain the process-developer’s responsibility to validate clearance for specific products and conditions.
Clearance obtained with the CHT/NaCl gradient system is as good as or better than that which is achieved with other chromatography techniques. In the context of its ability to simultaneously remove aggregates, leached protein A, DNA, endotoxins, and host cell proteins in combination with virus removal, however, CHT stands alone.
CHT’s resolution property makes it a powerful tool for process developers. The ability of CHT to purify a variety of proteins, including Mabs that are leading licensed products or therapeutic candidates in many drug companies, highlights its versatility. It is anticipated that CHT will gain even more favor as a component in purification processes.