Application note Supercoiled pDNA processing for consistent highpurification performance with a single platform for all sequence lengths Purifying plasmids for cell and gene therapy vectors requires chromatography systems where sequence size is not a limitation. This article describes a two step purification approach combining pDNAHERO® hydrophobic interaction chromatography and Q PuraBead® Edge anion exchange to deliver high-yield, high-purity plasmid DNA across a wide range of sequence lengths.
Multiple plasmids are necessary to manufacture raw materials used in cell and gene therapy, which can vary considerably in length. Lentiviral vectors are generated using a transfer plasmid, packaging plasmids, and an envelope plasmid; while AAV vectors require a helper plasmid, a RepCap plasmid, and a capsid plasmid. Size constraints inherent in poreor channel-based adsorbents mean purification of vector plasmids can be challenging. Often, separate chromatography media is required for longer sequences, particularly for those over 6 kilobases (kb).
Size-independent capture of pDNA
Convective flow
A robust pDNA purification platform will enable consistent performance independent of sequence length, ultimately supporting more reliable manufacturing of gene-based therapies. To provide consistent processing results where manufacturing demands are variable, Astrea Bioseparations has developed a two-step orthogonal purification process that enables efficient pDNA capture from large volumes of lysate and high resolution of supercoiled plasmid DNA (sc pDNA), with consistent performance for both short and long sequences. The capture step is performed with a hydrophobic interaction chromatography (HIC) adsorbent, pDNAHERO®, followed by polish with Q PuraBead® Edge anion exchange chromatography resin.
Screening through to process development
pDNA capture The pDNAHERO® addresses common bottlenecks in plasmid capture by replacing conventional pore- or channel-based chromatography media with an advanced electrospun nanofiber structure (Figure 1). Unlike packed resin beds, the interconnected flow paths of the nanofiber matrix eliminate dead zones where product can become trapped, and flow can be restricted. This novel architecture removes size and pressure limitations, enabling high-speed processing without sacrificing product recovery or purity.
Pilot-scale, GMP-compatible Figure 1: Purification of supercoiled pDNA is facilitated by pDNAHERO® and the convective flow of the AstreAdept® nanofiber matrix.