Disposable Process for cGMP Manufacture of Plasmid DNA - A low cost, disposable process for the manufacture of pDNA will aid in the development of vaccines. - BioPharm International


Disposable Process for cGMP Manufacture of Plasmid DNA
A low cost, disposable process for the manufacture of pDNA will aid in the development of vaccines.

BioPharm International Supplements


Figure 4
The purification step initially involves the diafiltration and concentration of the lysate with a hollow-fiber crossflow filtration cartridge. This device is assembled with a vacuum-resistant plastic container fitted with a series of three-way valves, vent filters, and Pharmed tubing in an enclosed system. The buffers, which are stored in bioprocess containers (BPCs), are attached to a three-way valve on the diafiltration line (Figure 4). An appropriate molecular weight cut-off membrane is chosen (300 kDa or smaller) to allow the retention and high recovery of supercoiled pDNA from the lysate. The SC nature of plasmid requires the selection of a membrane with a MW cut-off approximately 10 times smaller than expected to prevent losses across the membrane. Carefully defined operating conditions are also necessary to reduce the effect of shear, because plasmid is prone to affects that are exacerbated by the prevailing high salt conditions. The lysate is diafiltered, concentrated, and filtered into a BPC before anion exchange chromatography. Crossflow filtration of the lysate has been previously reported to be an excellent RNA removal step with more than 90% clearance demonstrated in practice (Figure 3).9

Anion exchange chromatography media in packed columns is the conventional approach for the initial capture of pDNA. The negatively charged pDNA molecules bind electrostatically to the media, but limitations in the binding capacity is a problem that remains unresolved, mainly because of the large size of the plasmid molecule. This has recently been addressed by a shift in emphasis from beaded media to a membrane approach. Membrane devices offer many benefits, including disposability (no need for column packing and qualification), an increased dynamic binding capacity for larger molecules, and high process flow rates.10

Figure 5
In our platform, a Mustang Q (Pall Corporation, East Hills, NY) capsule is used downstream from the crossflowed lysate (in contrast to other organizations that have used this step as a direct capture from E. coli lysate).11 Pretreatment by crossflow ultrafiltration is advantageous for increasing the Mustang Q's binding capacity for pDNA (since the RNA no longer competes once removed), improving recovery of eluted pDNA, and achieving higher product purities. The membrane device is attached to Pharmed tubing and three-way valves for processing during purification (Figure 5). The apparatus is autoclaved, operated in a Grade A laminar flow hood by a pump, and all fluid movements are contained in BPCs. If the step has been validated in terms of the wash conditions, elution volumes, and flow rates, the cartridge does not need to be attached to expensive instrumentation. The loading and washing of the sample is rapid, followed by a longer and staggered multiple high salt elution step. This ensures that the recoveries of pDNA achieve greater than 70% of total plasmid load, because membranes are characterized by slower off rates for pDNA.12 Mass balance analysis across this step efficiently removes 2 log10 orders of endotoxin and more than 50% of the remaining residual protein.

The eluate is collected into a BPC and ammonium sulphate is added to the BPC to achieve a final concentration of 1–1.5 M for the negative capture hydrophobic interaction step. At a set concentration of salt, impurities such as endotoxin (>90% removal) and RNA (>95% RNA removal) are selectively bound onto the HIC media while pDNA is recovered. The media is preconditioned with ammonium sulphate, autoclaved, and pumped into the BPC to achieve a specific nucleic acid ratio load to resin. The batch-mode HIC is incubated on a rotating mixer before 0.22 μM filtration into another BPC to remove the resin and recover the product.

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