Scale-Up of Plasmid DNA Downstream Process Based on Chromatographic Monoliths

Černigoj, Urh; Štrancar, Aleš

doi:10.1007/978-1-0716-0872-2_9

Cited by 9 publications

(14 citation statements)

References 24 publications

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“…The samples were dissolved in 10 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0 – both containing different pDNA isoforms, for the exact composition see Table 1. Plasmids were isolated from pDNA‐containing E. coli cell pellet (biomass) applying a procedure discussed elsewhere [18]. NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA) was used for quantification of pure pDNA samples (absorbance measurements at 260 and 280 nm).…”

Section: Methodsmentioning

confidence: 99%

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Pavlin,

Černigoj,

Bavčar

et al. 2023

Electrophoresis

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High‐performance liquid chromatography (HPLC)‐based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in‐process control monitoring of pDNA production processes.

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Section: Methodsmentioning

confidence: 99%

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Pavlin,

Černigoj,

Bavčar

et al. 2023

Electrophoresis

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“…pAAV2/8 (7.3 kbp), pKLAC1 (9.1 kbp), pGMAAV (11.6 kbp) and pAdDeltaF6 (15.4 kbp) were purified using HIP2 Plasmid Process pack (Sartorius BIA Separations, Ajdovščina, Slovenia) by optimising the manufacturer's protocol for each plasmid [12]. E. coli cell pellets were thawed in heated bath.…”

Section: Plasmid Isolationmentioning

confidence: 99%

“…The heart of downstream processing usually consists of one or more tangential flow filtrations (TFFs) and chromatographic steps [9,10]. Among different chromatographic supports, monolithic columns have already been successfully used in the industrial scale purification process of pharmaceutical grade pDNA due to their flow rate-independent separation and high dynamic binding capacity (DBC) [11][12][13]. The most standard choice for pDNA purification is anion exchange (AEX) chromatography, because DNA molecules are highly negatively charged, thus interacting with positively-charged AEX surface [14][15][16].…”

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confidence: 99%

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

Kralj,

Kodermac,

Bergoč

et al. 2023

Electrophoresis

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Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large‐scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion‐exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build‐up and column clogging during purification of plasmids at least up to 16 kbp in size.

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“…Purified pDNA for the method development was obtained from the pDNA-containing Escherichia coli cell pellet (biomass) using CIM HiP 2 Plasmid Process Pack 8 mL columns (Bia Separations, Ajdovščina, Slovenia) according to [19]. Briefly, biomass containing pDNA was resuspended in 50 mM Tris, 10 mM EDTA, pH 8.0.…”

Section: Pdna Isolationmentioning

confidence: 99%

Guanidine improves DEAE anion exchange‐based analytical separation of plasmid DNA

et al. 2021

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Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.

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Scale-Up of Plasmid DNA Downstream Process Based on Chromatographic Monoliths

Cited by 9 publications

References 24 publications

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

Guanidine improves DEAE anion exchange‐based analytical separation of plasmid DNA

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