DNA Removal By Depth Filters in Bioprocesses

Question By: Ashish Vishwakarma

Ans.: Dear Ashish, I would like to tell you that this is a Downstream Processing from Bioprocess purification Process in which gene coding for therapeutic proteins are inserted into host cell and fused with myeloma cell to produce hybridoma cell. these cell are grow in to the fermentation vessels in order to produce the desired therapeutics proteins.

Therapeutics proteins are transported in to culture medium by the host cell and purification begin with separation of the cell mass from the protein suspended in the culture medium. during these separation process host cell DNA can be release from dead or lysed cell.

eg. Zeta Plus depth filters

Process: DNA in final product dosage forms should not exceed 100 pg per dose. Host cell DNA can contaminate the culture fluid as a result of release by lysed or dead cells. In some cell culture clarification systems, centrifugation or tangential flow filtration (TFF) may be used as a primary clarification step. Both of these technologies are shear intensive and can increase cell lysis, resulting in increased host cell DNA contamination. The presence of contaminating DNA early in a process creates the possibility for carry through during downstream purification. It is therefore important to remove as much DNA as possible during the early stages of purification.

Fact: Zeta PlusTM depth filters, contain cellulose, filter aid, and a resin that imparts a positive charge to the filter surfaces. Zeta Plus depth filters are commonly used for initial clarification of cell culture broth and cell lysates. These filters contain significant voids volume (hence the name depth filter) that allow for accumulation of cellular debris. The filter structure is a series of interconnecting pore pathways able to retain cellular debris by mechanical entrapment. In addition to debris removal by mechanical retention, Zeta Plus filters are able to remove particles smaller than their pore size. This capability is based on electrokinetic attraction of negatively charged particles by the positively charged filter surfaces. It was anticipated that DNA, which is a polyanionic molecule, would also be removed by Zeta Plus filters.

CONCLUSION:

The removal of DNA from cell culture broths and lysates may be required to accomplish the CBER-recommended concentration of DNA impurity of <100 pg/dose in therapeutic dosage forms.

In many purification processes, it is desirable to remove impurities as early as possible from the process. 3M Purificationfs Zeta Plus depth filter medium has

been extensively used in upstream processes to clarify cell culture broths and cell lysates and serves as an effective means to reduce contaminating levels of DNA.

Reference: www.bioprocessintl.com/.../archive/.../BPI_A_100807AR23_O_103770a.pdf

Note: Dear ashish please patch your comment for further clarification