Neutralized E. coli cell lysate is a complex intermediate sample in the production process of plasmid DNA (pDNA) for use in vaccines and gene therapy. It contains various amounts of pDNA isoforms together with bacterial RNA and remains of bacterial genomic DNA, together with different ions (Na+, Ca2+, K+, NH4+, acetate, SO42- etc.) in millimolar to molar concentration range. These salts interfere with different analytical techniques, used for lysate characterization, thus decreasing method accuracy, sensitivity and/or robustness. One example of extremely matrix-dependent analytical technique is capillary gel electrophoresis (CGE) with LIF detection, which is often used for the analysis of pDNA isoform composition. Low sample conductivity is particularly essential for robust injection, which is not easily achieved for complex samples such as E. coli cell lysate. Currently, centrifugal filters with membranes of different materials and pore size cut-offs are used for sample desalting via ultrafiltration, where the degree of desalting is linked to the number of centrifugation cycles.
Here we present the use of an innovative method for desalting of E. coli cell lysate using monolithic spin columns and compare its performance with traditional centrifugal filters. In both cases, CGE was used for sample characterization after desalting. Utilizing the bind-elute mechanism, monolithic spin columns can efficiently remove unwanted salts as the column binds only nucleic acids, while salts pass through it. Under optimal conditions, samples prepared with monolithic spin columns retain the nucleic acid profile of the lysate, enabling precise analysis of nucleic acid/pDNA isoform composition in the E. coli lysate. Furthermore, one device and one general procedure can be used for rapid desalting of various nucleic acids (oligonucleotides, pDNA, RNA), regardless of their size, which is not attainable with centrifugal filters. Finally, the desalting procedure can also be transferred to CIMmultus column line and applied for preparative desalting of nucleic acids.
*The monolithic spin columns discussed in this study are not yet available for commercial sale.
