Purpose: Sodium Dodecyl Sulfate (SDS) is a critical reagent used in the purification of plasmid DNA for gene therapy applications. Potential contamination of SDS with nucleases presents a risk to the quality of the final purified product. Therefore SDS must be tested for the presence of nucleolytic enzymes. Several vendors certify the reagent as “DNase free”, however, this analytical testing does not take into account the fact that nucleases are reversibly inhibited by SDS itself. The purpose of this work was to develop a specific limit test method detecting potential nucleolytic activity in samples containing SDS.
Methods: Commercially available fluorogenic substrate (DNase Alert™ QC System) was used to detect nucleolytic activity in SDS samples spiked with DNase. Removal of SDS by several extraction techniques such as precipitation, solid phase extraction, and formation of mixed micelles was attempted.
Results: Our data confirmed earlier publications demonstrating reversible and dose-dependent SDS inhibition of DNase activity. Removal of SDS by precipitation or solid phase extraction was not reliable as it yielded less than 10% DNase spike recovery. Addition of Triton and Deoxycholate successfully neutralized SDS inhibition and reactivated DNase at 75% spike recovery.
Conclusion: The analytical limit test method was developed in support of DNase activity detection in the presence of SDS. The method was successfully validated as a limit test according to USP and is suitable for analysis of raw material used in the manufacturing process of clinical materials.
Elango Kathirvel– Senior Scientist, Nitto Avecia Pharma Services
Angela Nasseri– Manager, Nitto Avecia Pharma Services
Ming Li– Principal Scientist & Team Lead, Nitto Avecia Pharma Services
Aryo Nikopour– Senior Vice President, Scientific & Technical Services, Nitto Avecia Pharma Services