The column integrity test must be performed before first use and is the recommended test when column damage is suspected. Column integrity is evaluated with a pulse-response test, which measures the detector response (UV, conductivity) of a non-binding tracer. The tracer is injected into the flow path of the system and eluted by an isocratic elution with a suitable mobile phase. The duration from tracer injection to elution is the retention time of the tracer and should conform to a defined criterion. To account for dead volume of the system, it is necessary to first perform a tracer injection without connecting the column. A pulse response test is therefore performed on the LC system without the column and with the column connected to the system. The peak shape and the retention time of the tracer give an indication of column integrity.
HiP2 Plasmid Process Pack™ produces low endotoxin, highly homogeneous supercoiled plasmid DNA (pDNA) of clinical grade. The process is applicable for bench scale purification and can be scaled up for manufacturing of pDNA as raw material or drug substance. The advantage of monolithic columns allows high flow rates and short processing times.
Contaminants are removed without the use of enzymatic treatment. Potassium acetate and calcium chloride precipitation alongside two separate chromatography steps are used for removal of RNA, endotoxin, genomic DNA, protein and other contaminants. The first chromatography step separates contaminant RNA and proteins from the plasmid DNA. The polishing step eliminates remains of genomic DNA, endotoxin and open circular (oc) and linear pDNA isoforms, resulting in the isolation of the supercoiled (sc) pDNA isoform.