Quick Start Methods for Plasmid DNA Isoform Monitoring

21Jun2023

Plasmids (pDNA) are commonly used in molecular biology. They serve as carriers of genes for cell transfection in gene therapy and recently also as templates for in vitro mRNA production. Monitoring the isoform composition of pDNA is essential to assure quality of the final product.

Why it is important to monitor pDNA isoforms?

In both gene therapy and mRNA production, pDNA isoform composition is a crucial parameter in final characterization. It is important to monitor and understand the isoform profile of pDNA before entering the next process stages. Isoform monitoring is advised during the purification process of pDNA to identify important steps that can impact the integrity of supercoiled (sc) pDNA and potentially lead to the formation of open circular (oc) isoforms. This includes identifying sources of oc pDNA generation, such as shear-generating mixing modes and similar factors.

In mRNA production linearization of pDNA template is one of crucial steps and high linearization rate is favored. A good understanding of linearization kinetics during production ensures economical enzyme consumption and optimal step performance.

 Isoform Quantification and Separation with CIMac pDNA

 To obtain accurate and quantitative results, chromatography analytics with CIMac pDNA columns is highly recommended. These columns offer a fast and reliable solution for separating pDNA isoforms in just a 17-minute analysis. Samples are typically diluted with buffer and directly injected onto the column, simplifying the workflow while maintaining efficiency.

 Quick Solutions / Methods

For a comprehensive starting point in analyzing pDNA purity and isoforms, we provide a quick start guide. This guide offers detailed information on the suggested methods and buffers used. 

In addition to the quick start guide, we have developed an analytical platform package tailored for process development and pDNA production. This package provides "at-line" reliable insights by using a single system and method to quantify pDNA isoforms throughout the entire downstream processing workflow while ensuring the removal of impurities.