Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

Abstract

Within the biopharmaceutical industry, recombinant plasmid DNA is used both as a raw material (e.g. in lentiviral and AAV vector production) as well as an active ingredient (e.g. in DNA vaccines). Consequently, many analytical laboratories are routinely involved with plasmid DNA topoisoform qualitative analysis and quantification. In order to reliably determine plasmid topology, one must ensure that the methodology employed can reliably, precisely and accurately measure qualitatively and quantitatively all topological isoforms. Presented here are an anion-exchange high-performance liquid chromatography (AEC) and an agarose gel electrophoresis (AGE)-based method developed for this purpose. The strategies undertaken to overcome the respective typical problems of limited linear range of quantitation (for AGE) and isoform resolution (for AEC) are described. Also presented is a subsequent direct comparison (for assay precision/accuracy) of these two methods, as well as a package of species characterization [by chloroquine-AGE, enzymatic digestion, multi-angle laser light-scattering (MALLS) and electron microscopy] undertaken to confirm the identity of a minor supercoiled dimeric concatamer observed by both approaches.

Figure 1

Quantitative AGE assay development. (A) Ethidium bromide versus Sybr-Gold ‘Staining’. Aliquots of 200 ng of four plasmid batches known to contain differing levels of recombinant plasmid were separated by AGE and then stained with 1× TBE containing either ethidium bromide (50 ng/ml) (left gel) or 1× Sybr-Gold (right gel) for 30 min prior to gel-image capture by Polaroid camera. Arrows indicate the position of low-level recombinants only clearly visible with Sybr-Gold staining. (B) Image analysis of a 1× Sybr-Gold stained gel using manufacture's recommended staining times. Image capture by ProXpress. For this analysis, 1, 2 and 5 μl volumes of MassRuler High Range DNA Ladder (MBI-Fermentas, Lithuania) were loaded in lanes 1, 2 and 3, respectively. Left panel, gel image with loads corresponding to DNA quantities (per band) ranging from 1.6 to 10 ng (lane 1), 3.2 to 20 ng (lane 2) and 8 to 50 ng (lane 3). Middle panel, schematic ImageQuant (Molecular Dynamics, SunnyVale, CA) generated electropherogram traces of each lane. Right panel, scatter-plot with trend-line representation of area-under-curve signal (y-axis) against quantity (in nanograms) of DNA (x-axis) of the combined 1.6 ng through to 50 ng results. This plot highlights the significant signal ‘plateau’ that develops above 50 ng loads (C) Extending assay linear range by increasing Sybr-Gold staining times. Triplicate 5 μl (lanes 1, 3 and 5) and 30 μl (lanes 2, 4 and 6) MassRuler DNA ladder loads were separated by AGE. Subsequently, the gel was then dissected longitudinally into three parts and stained for 20 min (lanes 1 and 2), 2 h (lanes 3 and 4) or 24 h (lanes 5 and 6). After such staining regimes, excess stain was removed by copious washing with water. Image analysis by ProXpress and scatter plot data representation was then undertaken as described above in (B). The 5 and 30 μl loads generate a collective DNA species quantitative range of between 8 and 300 ng. The results of analysis reveal the R² values obtained for the 20 min, 2 h and 24 h staining regimes are 0.910, 0.990 and 0.999, respectively.

Figure 2

AEC assay development. Representative analytical traces generated by analysis of plasmid DNA using 2, 3, 4 and 5% gradients per minute as indicated (from left to right, respectively). Details of Buffer A (25 mM borate) and Buffer B (25 mM borate, 1 M NaCl) mixing parameters over time (in minutes) for each gradient are also included in the associated table. The flow rate (1 ml/min) and the column temperature (40°C) were constant for all analyses. For all gradients, the order of elution from open-circle (OC, first off the column) through to fraction D (D, last off) was maintained. However, note that for a 2% gradient, complete separation of supercoiled (SC), linear (L) and fraction D (D) was not possible. The retention times of all known peaks correlated with the enzymatically prepared reference standards (data not shown). For characterization of the unknown peak see Figures 5 and 6 (fraction D).

Figure 3

Results from a Q-AGE and AEC direct assay comparison comprising two samples analysed in triplicate on three separate occasions. (A) Example gel image obtained by AGE (by 24 h Sybr-Gold staining methodology). Triplicate 200 ng loads of purified plasmid (lanes 1–3) and 37°C stored purified plasmid (lanes 4–6) were analysed. The position of the supercoiled (SC), linear (L) and open-circle (OC) species are indicated. The schematic longitudinal tracks through samples 1 and 4 are a representation of the cross sections subsequently analysed by ImageQuant. (B) ImageQuant generated schematic traces obtained from the cross sections of lanes 1 and 4 seen by AGE. (C) The corresponding AEC schematic traces obtained from the two different samples. (D) Tabulated results of AGE and AEC direct comparison. For each sample type (purified plasmid or temperature-induced degraded plasmid), the values for the mean and relative standard deviation (RSD) of triplicate runs on three separate occasions are presented (i.e. nine values per mean result). The RSD value obtained (in parentheses) can be considered representative of total intra-assay precision (repeatability).

Figure 4

A direct assay comparison: Stability study samples analysed by AGE- and HPLC-based methodologies. (A) Gel image of the −20, +4 or +37°C stored samples separated by 0.6% AGE. Lane order: −70°C stored purified plasmid reference standard (ref), time zero (lane 0), 1 month time point (lane 1), 2 month time point (lane 2), 3 month time point (lane 3), 4 month time point (lane 4), 5 month time point (lane 5), 6 month time point (lane 6). The positions of the supercoiled (SC), linear (L) and open-circle species (OC) are indicated. (B) Line plot of stability study results as analysed by AGE methodology. Relative proportions of each topological sub-species (x-axis) over time (y-axis) are presented. Also included are numerical results at the final 6 month time point (in parentheses). (C) Line graph plot of stability study results as analysed by HPLC methodology. Relative proportion of each topological sub-species (x-axis) over time (y-axis) are presented. Also included are numerical results at the final 6 month time point (in parentheses).

Figure 5

Analysis of different topological plasmid isoforms by AGE- and AEC-based methodologies. (A) Analysis of equivalent amounts of differing forms by both methodologies. One representative image of the triplicate AGE-based analyses undertaken is presented. The predominately linear (lane 1), open-circle (lane 2) and supercoiled (lane 3) plasmid species isoforms were prepared as described in the text. AEC analysis was also undertaken in triplicate. Also presented are the tabulated results of the mean data values obtained by such analysis by both methods. These results are offered as both absolute raw assay data values and also signal strength relative (in %) to the predominantly supercoiled parental control. Results indicate that by AEC-based methodologies, the levels of the open-circle species observed are reduced. (B) Analysis of a linear/open-circle ‘spike’ preparation by both methodologies. Triplicate analyses of an AEC equated 10% linear/open-circle spiked plasmid (spiked according to a prior AEC-based abs260 quantification of starting material) was undertaken by both methods. A representative image generated during AGE analysis is shown. Also presented are the tabulated results of AGE- and AEC-based analysis. These results demonstrate that whilst open-circle levels in an AEC equated 10% spike preparation are recorded as ∼10% by subsequent analysis, such samples report much greater levels of open-circle by AGE.

Figure 6

Fraction D analysis by AGE. (A) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. (B) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. (C) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. (D) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. (E) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).

Figure 7

Physical characterization of differing plasmid isoforms. (A) Analysis by EM. The four major fractions observed by AEC were collected separately (i.e. purified) and then analysed. Representative images for all four fractions are shown as indicated and correlate with previous work, suggesting that they represent (in order of elution) open-circle (OC), supercoiled (SC), linear (L) and supercoiled dimer, respectively. Note that minor Adobe image contrast adjustments were employed to aid in visualization. (B) Analysis by MALLS. All fractions were analysed and the weight and size data (reported relative to the supercoiled fraction) are presented. Results suggest fraction D to be nearly twice the molecular weight of the open-circle, supercoiled, linear fractions, thus being in agreement with fraction D being a dimeric species.