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Sample displacement chromatography of plasmid DNA isoforms
Journal of Chromatography A, 1414 (2015) 103–109
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Sample displacement chromatography of plasmid DNA isoforms a,∗ ˇ Urh Cernigoj , Urˇska Martinuˇc a , Sara Cardoso a,b , Rok Sekirnik a , Nika Lendero Krajnc a , a,c ˇ Aleˇs Strancar a
BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovˇscˇ ina, Slovenia University of São Paulo, Escola Politécnica da USP – Department of Chemical Engineering, Av. Prof. 7 Lineu Prestes, 580, Conjunto das Químicas, Bloco 18, 61548, CEP 05424-970 São Paulo, Brazil c The Centre of Excellence for Biosensors, Instrumentation and Process Control – COBIK, Velika pot 22, SI-5250 Solkan, Slovenia b
a r t i c l e
i n f o
Article history: Received 10 May 2015 Received in revised form 13 August 2015 Accepted 14 August 2015 Available online 20 August 2015 Keywords: Plasmid DNA Hydrophobic interaction chromatography Chromatographic monoliths Preparative chromatography
a b s t r a c t Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1 mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Displacement chromatography (DC) is a chromatographic technique that depends on the use of three mobile phases: a carrier, a displacer and a regenerant [1]. The carrier facilitates the load by solubilising the sample, and enables positive interaction with the stationary phase. The displacer leads displacement of the sample components from the stationary phase; during this step, individual sample components may also displace each other from binding sites on the stationary phase. Finally, the regenerant removes the displacer from the stationary phase and regenerates the column for the next chromatographic cycle [2]. DC offers several advantages over traditional chromatographic techniques, such as effective separation factors between feed solutes and high sample loading,
∗ Corresponding author. ˇ E-mail address: [email protected] (U. Cernigoj). http://dx.doi.org/10.1016/j.chroma.2015.08.035 0021-9673/© 2015 Elsevier B.V. All rights reserved.
leading to enhanced productivity [1–4]. It is usually performed in ion-exchange [5] or hydrophobic interaction chromatography (HIC) mode [6]. The development of low molecular mass displacers for protein purification in ion exchange [7] and HIC systems [6] offers the advantage of ease of separation of the displacer from purified proteins using size-based purification methods. When the displacer is itself a component of the feedstream, the method is known as sample displacement chromatography (SDC). The method takes advantage of different relative binding affinities of sample mixture components, such that during sample loading, the components compete for adsorption onto a stationary phase. SDC approach should optimally be performed under overloading conditions, since the main separation occurs already during sample loading and column capacity is thus optimally used [8]. SDC was introduced for preparative purification of peptides in reverse-phase mode [9] and has subsequently been successfully applied for purification of proteins in ion-exchange [10], affinity [11] and HIC [12] modes, using traditional porous particle chromatographic media,
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operating under diffusion mass transfer conditions. SDC approach is most useful for samples where the product binds to a chromatographic support more strongly than the impurities, because this enables fast and efficient purification of complex samples [8,11]. Due to convective mass transfer which leads to diffusionindependent chromatographic properties, the use of convective chromatographic supports, such as monoliths, is advantageous for performing DC and SDC purification of biomacromolecules over particulate chromatographic supports [10,13]. For successful SDC on particulate chromatographic supports, flow rates must be five times lower to achieve optimal sample displacement compared to standard gradient elution separation [14]. On the contrary, flow rate-independent SDC for purification of monoclonal antibodies has been demonstrated with convective media [15]. Since SD effects on monoliths are independent of column size [10], they can be used for SDC of biomacromolecules at microanalytical, analytical and preparative scales. Despite the increasing pharmaceutical potential of plasmid DNA (pDNA), which is typically purified using step gradient elution [16,17], only a single study applied DC/SDC approach to its purification [18]. The study demonstrated that conventional porous particle columns were not suited to the separation of any two substances varying considerably in molecular mass, whereas of separation of pDNA from host cell proteins (HCP) and endotoxin was achieved using a continuous bed column. Pharmaceutical-grade pDNA requires high homogeneity of supercoiled (sc) form, and chromatographic separation of sc from open coiled (oc) or linear isoforms on preparative scale is extremely challenging due to their structural similarity [19]. DC/SDC approaches have not yet been investigated for the separation of pDNA isoforms, and the main purpose of the present work was to develop a method for separation of sc pDNA from other pDNA isoforms using SDC on monolithic chromatographic supports, that would enable high resolution, high capacity and scalable pDNA purification methods in the future. 2. Materials and methods 2.1. Materials All solutions were freshly prepared using purified water which meets the requirements for European Pharmacopoeia (AQUATEHNA Biro, Zgornja Kungota, Slovenia) and analytical grade reagents. Buffer solutions were filtered through a 0.22 m PES filter (TPP, Trasadingen, Switzerland). Agarose gel was prepared from SeaKem LE Agarose (Lonza Group, Basel, Switzerland). Sodium hydroxide (NaOH), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), acetic acid, bacto tryptone, yeast extract, potassium acetate, calcium chloride (CaCl2 ), sodium chloride (NaCl), ammonium sulfate (AS), sodium dodecyl sulfate (SDS), 2-mercaptopyridine and hydrochloric acid (HCl) were from Sigma–Aldrich (St. Louis, MO, USA), EDTA was from Kemika (Zagreb, Croatia) and boric acid was from Merck (Darmstadt, Germany). 1 ,1 -carbonyldiimidazol (CDI) was purchased from Tokyo Chemical Industry (Tokyo, Japan), while histamine hydrocloride from AppliChem (Darmstadt, Germany). 2.2. Chromatographic equipment and columns All chromatographic experiments were carried out using a gradient chromatography workstations, consisting of two pumps, optionally an autosampler with various sample loop volumes and a UV detector – Smartline (Knauer, Berlin, Germany), Äkta Purifier (GE Healthcare Life Science, Uppsala, Sweden) and model HP 1200 Series chromatograph (Agilent Technologies, Santa Clara, USA). For data acquisition and control, EuroChrom 2000 software (Knauer),
Unicorn (Äkta Purifier) and ChemStation (Agilent Technologies) were used. All CIMac monolythic analytical columns (V = 0.106 mL) were provided by BIA Separations (Ajdovˇscˇ ina, Slovenia). Three different types of hydrophobic columns were used for the preparative loadings: C4 HLD, pyridine and histamine modified columns. CIM C4 HLD is a commercialised product of BIA Separations with a butyl ligand density of 1.8 mmol/mL of the support. CIM-pyridine monolith was prepared by modifying the CIMac epoxy monolith with an ethanolic solution of 2-mercaptopyridine. The achieved pyridine ligand density on the chromatographic support was 1.0 mmol/mL, determined from elemelemental analysis. Histamine was immobilised on CIMac CDI monolith leading to the neutral carbamate bond, as it is described thoroughly in reference [19]. 2.3. Cell lysis protocol and plasmid purification All experiments were conducted using two different plasmid vectors: pEGFP–N1 (4700 bp) and pMD204 (2345 bp). The pEGFP–N1 (4700 bp) was provided by DSMZ (Braunschweig, Germany). Liquid cultures were cultivated overnight at 37 ◦ C on Luria-Bertani broth containing appropriate antibiotics. Bacterial biomass from Escherichia coli strain DH5␣ containing plasmid vector pMD204 (2345 bp) was obtained from the Faculty of Chemistry and Chemical Technology (University of Ljubljana, Slovenia) [20]. Bacteria were lysed using lysis procedure according to Smrekar et al. [21]. Chromatographic purification of pDNA from clarified lysate was performed using CIMTM HiP2 Plasmid Process Pack 8 mL columns (BIA Separations, Ajdovˇscˇ ina, Slovenia) according to manufacturer’s instructions. Samples for the calibration curve and for the enzymatic restrictions were additionally treated with Amicon® Ultra centrifugal filters (10,000 MWCO) (Millipore, Cork, Ireland) to exchange buffer to 20 mM TRIS, pH 7.4 and to concentrate the pDNA concentration up to 1.2 mg/mL. Oc pDNA was prepared from the sample containing sc pDNA in 50 mM TRIS, 10 mM EDTA, pH 7.4, by exposure of the sample to 60 ◦ C for 48 h. Linear isoform was prepared enzymatically using restriction endonuclease PstI in the buffer and conditions recommended by the supplier (Thermo Ficher Scientific, Waltham, USA). 2.4. Chromatographic experiments in buffers containing high concentrations of AS All chromatographic runs were carried out on CIMac 0.106 mL columns. Analytical runs were performed in AS gradient at the flow rate of 1.0 mL/min, where the buffer A was 50 mM TRIS, 10 mM EDTA, 3.0 M AS, pH 7.4 and the buffer B 50 mM TRIS, 10 mM EDTA, pH 7.4. 500 l of pEGFP sample of different concentrations and isoform compositions was injected on the column followed by a 25 CV gradient to 100% buffer B. The selectivity (˛) between pDNA isoforms was calculated according to Eq. (1), where VR,sc and VR,oc are the elution volumes of sc and oc pDNA isoforms in mL, while V0 is the void volume of the column in mL. ˛=
(VR,sc − V0 ) (VR,oc − V0 )
(1)
For preparative chromatography, pDNA samples of different concentrations and isoform compositions were prepared in a buffer containing 50 mM TRIS, 10 mM EDTA and various concentrations of AS, pH 7.4. Frontal analyses experiments were then performed for each sample at different flow rates and pDNA loading was monitored using UV absorbance at 260 nm.
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2.5. pDNA analysis 2.5.1. Agarose electrophoresis (AGE) Chromatography fractions containing pDNA were analysed by horizontal AGE (Takara Bio, Inc, Otsu Shiga, Japan) using SYBR Safe DNA gel stain (Invitrogen, Eugene, VI, USA), 6 × Orange DNA Loading Dye (Thermo Ficher Scientific, Waltham, USA) and GeneRuler DNA Ladder Mix (Thermo Ficher Scientific, Waltham, USA). Running buffer was 1 × TBE (40 mM TRIS, 20 mM boric acid, 1 mM EDTA, pH 8.0). Gel was prepared with 0.7% agarose in 1 × TBE buffer with 1 × SYBR Safe DNA Stain. 2.5.2. Analytical chromatography HPLC analyses were conducted on an Agilent HP 1200 Series chromatograph, coupled with a multiwavelength detector. Chromatographic separations were run on a CIMacTM pDNA analytical column (BIA Separations, Ajdovˇscˇ ina, Slovenia) with bed volume of 0.318 mL. The equilibration buffer consisted in 200 mM TRIS, pH 8.0, whereas the elution buffer was 200 mM TRIS with 1.0 M NaCl, pH 8.0. The flow-rate used was 1.0 mL/min and the gradient consisted of: equilibration buffer for 1 min, linear gradient from 0% to 60% of elution buffer (0.6 M NaCl) in 1 min, 1 min hold at 60%, a linear gradient from 60% to 70% of elution buffer (0.7 M NaCl) in 10 min, and a step gradient to 100% of elution buffer. The absorbance was measured at 260 and 280 nm. Quantification of pDNA was performed using a calibration curve with pure plasmid samples of known concentrations. A serial dilution of pDNA (between 1.0 and 120 g/mL) was prepared by diluting a stock solution (1 mg/mL) with 200 mM TRIS, pH 8.0. All pDNA concentrations used for calibration curve were determined spectrophotometrically with a Smart Spec 3000 spectrophotometer (Bio-Rad, Richmond, USA). One unit of OD 260 nm in a 10 mm cuvette is assumed to correspond to 50 g/mL of a double stranded DNA. Concentration obtained by spectrophotometer was correlated with the corresponding peak area on the chromatogram obtained by CIMac pDNA analytical column. 3. Results and discussion 3.1. Sample displacement of oc pDNA by sc pDNA It has previously been demonstrated that pDNA isoforms may be separated preparatively using CIM monoliths [21] in HIC mode. The process reported used a C4 high ligand density (HLD) CIM monolith column in conjunction with loading at 3.0 M AS, washing with intermediate (1.6 M) AS to remove oc pDNA isoform and finally elution of sc pDNA with low (0.4 M) AS. Although pDNA isoforms have been successfully separated, the drawback of the described process is the high molarity of AS required, thus increasing the cost of goods for preparative purification of pDNA. This study therefore attempted to use SDC approach to decrease the AS concentration required
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during loading onto hydrophobic chromatographic supports. Optimal AS concentration range was determined by analytical separation of two pDNA isoforms in a negative AS concentration gradient (HIC conditions). Three different ligands immobilised onto CIMac monolithic support were tested: C4 HLD, pyridine and histamine. AS elution concentrations for both pDNA isoforms, column efficiency (expressed as width of each chromatographic peak = WB ) and selectivity (˛) were calculated (Table 1). Column efficiency and selectivity, rather than resolution, were parameters used for the evaluation of pDNA isoform separation efficiency, because the contribution of each parameter on SDC could be assessed separately. While all three chemistries demonstrated similar AS elution concentration for sc isoform (between 1.54 M for C4 HLD and 1.70 M for pyridine), they differed considerably in AS elution concentration for oc isoform (between 0.82 M for C4 HLD and 1.60 M for histamine). CIM-histamine demonstrated the highest efficiency and the lowest selectivity (WB for sc pDNA 1.6 min; ˛ 1.07), C4 HLD the highest selectivity and the lowest efficiency (WB for sc pDNA 6.6 min; ˛ 1.88) and CIM-pyridine demonstrated intermediate selectivity and efficiency (WB for sc pDNA 3.0 min; ˛ 1.29). Next, dynamic binding capacity (DBC) at 10% breakthrough measurements for oc as and sc pDNA isoform standards were determined in a loading buffer containing 1.75 M AS (Table 1). DBC values at constant loading conditions correlate with the strength of interaction between analyte and chromatographic surface. As expected the capacities are low for oc isoform (0.007–0.13 mg/mL) due to weaker interactions with the monolithic surface in 1.75 M AS, while the DBC values for sc pDNA differed depending on the hydrophobicity of ligand used: highest DBC (1.61 mg/mL) was observed for CIM C4 HLD (most hydrophobic matrix), whereas the lowest DBC (0.03 mg/mL) was observed for CIM-histamine matrix with lowest hydrophobicity. Another reason for larger DBC values for sc pDNA can be explained by the high supercoiled degree of sc isoform that enables the reduction of the superficial contact area, resulting in more dense packing of the plasmid onto a surface [22]. In order to prove this hypothesis, a loading of each isoform should be performed at the conditions, where binding energy for both isoforms is high enough (i.e. in 3 M AS) to achieve a chromatographic surface limited and not pDNA-monolith interaction limited DBC for each pDNA isoform [23]. We continued with the frontal analyses experiments of the mixture of pEGFP isoforms (20% oc pDNA and 80% of sc pDNA) on C4 HLD, histamine and pyridine-based supports. Three different AS concentrations in loading buffer were tested for each column. Three typical chromatograms (one for each chromatographic support) are shown in Fig. 1 while the calculated data from all 9 experiments are collected in Appendix (Table A.1) and Fig. 2. A clear double breakthrough is present in 6 of 9 experiments. AGE analysis suggested that the first breakthrough and ensuing plateau correspond to oc isoform breakthrough. The second breakthrough corresponds to sc pDNA isoform appearing together with
Table 1 Chromatographic characterisation of three different CIMac hydrophobic columns for pDNA isoforms separation. Column chemistry
cAS (oc pDNA) (M)
cAS (sc pDNA) (M)
˛
WB (oc pDNA) (min)
WB (sc pDNA) (min)
DBC50 (oc pDNA) (mg of pDNA/mL of the support)
DBC50 (sc pDNA) (mg of pDNA/mL of the support)
C4 HLD pyridine histamine
1.54 1.70 1.69
0.82 1.32 1.60
1.50 1.30 1.07
6.2 1.7 1.2
6.6 3.0 1.6
0.13 0.05 0.007
1.61 0.53 0.03
Columns 2–6: Separation of oc and sc pDNA mixture in AS descending gradient under analytical loading conditions. The AS elution concentration, selectivity (˛) and base width (WB ) of chromatographic peaks for oc and sc pEGFP-N1 isoforms on different CIM hydrophobic supports. Column volume (CV) = 0.106 mL, Q = 1.0 mL/min, = 260 nm, Vinj = 50 l, Sample: pDNA (20 g/mL) in buffer A. Buffer A: 50 mM TRIS, 10 mM EDTA, 3 M AS, pH 7.4; Buffer B: 50 mM TRIS, 10 mM EDTA, pH 7.4. Gradient from 0 to 100% buffer B in 47 CVs. Columns 7–8: DBC10 values for pure oc and pure sc pEGFP isoforms on three different chromatographic columns in a loading buffer containing 1.75 M AS. CV = 0.106 mL, Q = 0.5 mL/min, = 260 nm, Loading: sc pDNA (20 g/mL) or oc pDNA (5 g/mL) in 50 mM TRIS, 10 mM EDTA, 1.75 M AS.
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Fig. 1. Loading of a mixture of oc and sc pDNA (pEGFP) with sc pDNA homogeneity of 80% on different CIMac columns in buffers containing specific concentrations of AS. Loading buffer: 50 mM TRIS, 10 mM EDTA, different concentrations of ammonium sulfate (AS), pH 7.4. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4; Q (volumetric flow rate) = 0.5 mL/min, = 260 nm. Pyridine column: (oc pDNA) = 3.5 g/mL, (sc pDNA) = 15 g/mL, c (AS) = 2.0 M. C4 HLD column: (oc pDNA) = 4.7 g/mL, (sc pDNA) = 19.8 g/mL, c (AS) = 1.7 M. Histamine column: (oc pDNA) = 4.6 g/mL, (sc pDNA) = 19.0 g/mL, c (AS) = 2.1 M.
high concentrations of oc pDNA. Sc pDNA isoform with higher homogeneity compared to the load was detected in all elution fractions. The observation of a double breakthrough correlates well with the different DBCs for pure pDNA isoforms on different columns (Table 1). However, the appearance of a double breakthrough alone does not indicate a sample displacement (SD). SD can only be inferred by comparison of the oc pDNA concentration in the second breakthrough with its concentration in the load (Fig. 2A), where the ratio should be higher than 1. pDNA isoform concentration was quantified by CIMac pDNA analysis.
A higher concentration of oc in breakthrough compared to the load was observed for CIM-pyridine and CIM-histamine columns, suggesting that SD effect is in operation (Fig. 2A). The effect was more pronounced at higher AS concentrations: when loading concentration of AS was lowered to the proximity of the elution concentration for oc pDNA, displacement was not observed. In such case, a double breakthrough resulted mainly from the lower DBC for oc isoform. When the loading AS concentration was increased, displacement of oc molecules was observed. Effect differed for different types of hydrophobic monoliths; C4 HLD column showed a very limited SD effect regardless of the AS concentration used, likely because of a higher selectivity and broader chromatographic peaks (sc pDNA cannot displace oc pDNA because of the binding energy difference). In contrast, CIM-histamine column, where the selectivity is lowest and efficiency is highest, the SD effect was most pronounced. The results raised the possibility of extending the applicability of SDC for the removal of other nucleic acid-based impurities in pDNA downstream process, such as RNA or gDNA. Future studies should focus on extending the SDC approach to these molecules as well. 3.2. Purification efficiency of sc pDNA Since the main purpose of the work was the purification of sc from oc pDNA, it was necessary to analyse the elution fractions for all experiments as well. The sc pDNA homogeneity increased with the decrease of AS loading concentration (see Fig. 2B) for all ligand chemistries. It is speculated that a thermodynamic equilibrium between adsorbed sc and oc pDNA molecules is formed at each loading chromatographic conditions and with lowering AS concentration this equilibrium is shifted towards higher content of sc pDNA isoform. Purification of sc pDNA isoform at certain chromatographic conditions cannot exceed the thermodynamic equilibrium ratio. The correlation between the SD effect and homogeneity of the sc pDNA in the elution fraction is not straightforward. The more pronounced SD effect does not necessarily result in better homogeneity. Higher was the selectivity, less pronounced was SD effect,
Fig. 2. The detailed analysis of sample displacement effect – influence of AS concentration as well as type of the chromatographic support. A – ratio between oc pDNA concentration in breakthrough vs. its concentration in load; B – homogeneity of sc pDNA in the elution fraction. The figure is based on the evaluation of the nine different frontal analyses experiments, three of them are shown in Fig. 1 pDNA isoforms quantification in different fractions were performed using CIMac pDNA column as described in Section 2.5.2. Additional information on the experiments is included in Appendix (Table A.1).
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but an excellent homogeneity could still be obtained (for example around 98% for C4 HLD monolith). In such case, the oc pDNA was mostly removed by negative chromatography mode (the oc pDNA isoform did not bind to the matrix). On the contrary, the histamine column showed highly expressed SD effect even in 2.4 M AS loading buffer, but the sc pDNA homogeneity in the eluted fraction was just slightly improved compared to the load. At such high concentration of AS both isoforms bound much stronger to the matrix, and consequently the removal of oc form of pDNA by negative chromatography mode at such conditions became negligible and the oc isoform cannot be sufficiently removed solely by SD. Another important aspect of preparative chromatography is the amount of the purified analyte in the elution fraction. Up to 1.7 mg per mL of monolith support, the amount of eluted pDNA increased with the increase of AS concentration in the load, regardless of the chemistry used (Supporting information Table 1). Increasing the concentration of AS further would likely result in elution of pDNA at the limit of binding capacity for hydrophobic CIM monolith with average pore size diameter of 2100 nm (3 mg/mL of support) [23]. However, the homogeneity of sc pDNA in elution fractions was below 95% at the highest AS concentrations used with each specific support in this study. Therefore, when purifications are performed in SDC mode, the optimal loading AS concentration should be defined by the balance between the binding capacity and homogeneity (purity) of the final product.
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Fig. 3. Agarose electrophoresis (AGE) picture of SDC samples from mixtures of three pDNA isoforms on CIMac pyridine column. The bands from 1 to 11 on AGE gel correspond to fractions at different times during loading, presented in the inserted chromatogram. Band L is a loading fraction, while band E is an elution fraction from the column (the elution is not shown on the inserted chromatogram). Q = 0.5 mL/min, = 260 nm, (oc pDNA) = 3.0 g/mL, (lin pDNA) = 5.0 g/mL, (sc pDNA) = 13 g/mL in 50 mM TRIS, 10 mM EDTA, 1.90 M AS, pH 7.4. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4.
3.3. SDC for different plasmids In all subsequent experiments CIM-pyridine column was used at the optimal binding conditions (the amount of adsorbed plasmid and sc pDNA homogeneity) derived above. In order to demonstrate general applicability of monolithic columns for SDC of pDNA, purification was performed on a representative plasmid (pMD204, 2347 bp) (see Figure A.2 in Appendix). AS concentration in binding buffer depends on the hydrophobicity of individual pDNA molecule (influenced by base pair type composition and on the size of the pDNA [23]) and has to be determined for each pDNA sample separately. For isoform separation of pMD204 with CIM-pyridine optimal concentration of AS in loading buffer was determined at 2.0 M. The initial sample contained 30% of oc isoform; pDNA isoform quantification suggested that that 2.0 M AS led to a ratio of oc pDNA concentration in the second breakthrough vs. oc concentration in the load of 1.17, confirming displacement of oc by sc pDNA. Additionally, the homogeneity of sc isoform in elution fraction was 98%. The amount of eluted pDNA was 1.5 mg/mL of column.
Fig. 4. pDNA (pEGFP) loading and a double breakthrough under SDC conditions of samples containing different ratios between oc and sc pDNA isoform. CIMac pyridine column: (sc + oc pDNA) = 23 g/mL in 50 mM TRIS, 10 mM EDTA, 1.95 M AS buffer, pH 7.4, Q = 1.0 mL/min, = 260 nm. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4.
3.5. The effect of different oc:sc pDNA ratios in loading sample on SDC 3.4. Sample displacement of linear isoform Next, a removal of linear DNA isoform of pEGFP plasmid was analysed under SDC conditions. AS elution concentration for linear isoform was 1.68 M, similar to oc pDNA isoform (1.70 M), therefore it was concluded that SDC could work for removing linear isoform as well. A mixed sample (in 1.9 M AS) was therefore prepared containing 30% of linear isoform, 10% of oc isoform and 60% of sc isoform, and loaded on the pyridine column under SDC conditions. AGE analysis (Fig. 3) suggested that the first breakthrough correlated with the appearance of linear and oc isoforms, while the second breakthrough corresponded to sc pDNA, suggesting the enrichment of sc isoform in the elution fraction. pDNA analysis revealed a ratio of linear isoform concentration in fractions 7 and 8 vs. linear isoform concentration in load as 1.2 (Fig. 3), confirming a displacement of linear isoform by sc isoform. Additionally, the homogeneity of sc isoform in elution fraction was 98%, meaning that the enrichment from load to elution was 1.6 (Fig. 3).
In order for the pDNA purification method to be useful on preparative scale, it must apply to different oc:sc pDNA mixtures. It is known for pDNA purification using gradient elution that the AS concentration in wash buffer for oc pDNA has to be optimised for different oc:sc pDNA ratios [23]. It was speculated that SDC should be independent of the pDNA isoform ratio, because it is applied at overloading conditions. The SDC method was therefore tested on samples containing 10, 25 and 50% of oc isoform (Fig. 4). The concentration of AS in load was 1.95 M. The appearance of a double breakthrough was most evident for the sample containing 50% oc pDNA, while it was not strongly pronounced for the sample containing 10% oc pDNA. However, analysis of oc pDNA concentrations in different fractions suggested that SD operated for samples with 10 and 25% oc isoform (see Table 2), while for 50% oc isoform the concentration of oc pDNA in load and in the displaced fraction was very similar resulting in small relative difference. The homogeneity of the sc pDNA in the elution fraction
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Table 2 pDNA isoforms analysis from preparative SDC chromatography – pEGFP loading of samples containing different ratios between oc and sc pDNA isoform on CIMac pyridine column. % of oc isoform in load
Ratio between oc pDNA concentration in breakthrough fraction and in load
Amount (mg) of eluted pDNA per mL of column
sc pDNA homogeneity in elution fraction
10 25 50
1.10 1.21 1.05
1.12 1.15 1.09
97 96 95
The loading buffer contained 1.95 M AS. FT1, FT2 and FT3 fractions correspond to samples indicated on the chromatograms in Fig. 4.
4. Conclusions Sample displacement chromatography (SDC) of pDNA isoforms under HIC conditions has been demonstrated for the first time. Hydrophobic columns with different affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under overloading conditions and the separation efficiency was shown to be dependent on the selectivity of the column for different isoforms as well as on column efficiency. The SD effect often overlaps with non-binding (negative mode) effect, additionally influencing the separation efficiency. The loading AS concentration influences the purity of the elution fraction, obtaining higher purity with lower AS concentrations. The SDC was successfully implemented for the enrichment of sc pDNA for differently sized plasmid vectors, for the separation of linear isoform from sc isoform and for samples containing different oc:sc isoform ratios (even at 1:1 ratio). Like other chromatographic processes involving CIM monoliths, SD efficiency is flow rate-independent, and has been demonstrated at linear velocity of 450 cm/h. The potential of this method could be found in the preparative chromatography of pDNA because of several advantages over step gradient elution mode, such as a lower concentration of AS required during loading and removing a chromatographic step (wash) during the chromatographic process. Although DBC for pDNA is lower in SDC compared to standard pDNA purification methods, this can be avoided by utilising continuous, multicolumn chromatography systems, with which SDC is compatible, and which should be addressed in future studies. Acknowledgements
Fig. 5. SDC of pEGFP isoforms at different flow rates. CIMac pyridine column: (oc pDNA) = 3.6 g/mL, (sc pDNA) = 14.8 g/mL in 50 mM TRIS, 10 mM EDTA, 2.0 M AS buffer, pH 7.4, = 260 nm. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4.
decreased from 97 to 95% with the substantial increase of the initial oc:sc ratio. However, even at 1:1 ratio of oc:sc isoforms the enrichment factor of sc isoform in the eluted pDNA fraction was very high (1.9), suggesting the ability of monoliths to purify very heterogeneous pDNA samples to pharmaceutical grade in a single step. The possibility of using the same concentration of AS in binding buffer for different oc:sc isoform ratios makes the SDC more advantageous (robust) compared to gradient elution pDNA purification. 3.6. SDC at different flow rates One of the unique features of monolith chromatographic columns is flow-rate independent separation that was demonstrated for SDC of proteins [24], but had never been shown for plasmids. Therefore, separation of pDNA isoforms (80% homogeneity) under HIC conditions was tested at 2.5 CV/min (75 cm/h), 5 CV/min (150 cm/h) and 15 CV/min (450 cm/h) flow rates (Fig. 5). The x-axis of the loading chromatograms was unified to mL in order to compare the effect of different flow rates. Elution profile, when standardised for CV/min was identical for all flow rates (Fig. 5), suggesting that SDC operates at flow rates up to 15 CV/min (450 cm/h). DBC values of 1.30 ± 0.1 mg/mL were obtained for all flow rates used, consistent with convective mass transfer of molecules within the monolithic structure [23]. Importantly, the homogenity of the sc pDNA (approx. 97.5%) in the elution fraction was not affected by the flow rate, suggesting also that shear forces are not a limiting factor when using monolithic supports at very high flow rates.
We are greatly thankful to prof. Dr. Marko Dolinar from the University of Ljubljana, Faculty of Chemistry and Chemical Engineering, for providing us a pMD204 sample. Urˇska Vidic, Sandra Kontrec and Miroslava Legiˇsa from BIA Separations are acknowledged for their help in laboratory work. This work was partially financially supported by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia within the research programme P4-0469. Support of this research by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 312004 is also gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.08. 035 References [1] J. Frenz, C. Horvath, High-performance displacement chromatography, in: C. Horvath (Ed.), High Performance Liquid Chromatography – Advances and Perspectives, 5, San Diego Inc., San Diego, 1988, pp. 212–314. [2] H. Kalasz, Displacement chromatography, J. Chromatogr. Sci. 41 (2003) 281–283. [3] C. Horvath, A. Nahum, J.H. Frenz, High-performance displacement chromatography, J. Chromatogr. 218 (1981) 365–393. [4] R. Freitag, J. Breier, Displacement chromatography in biotechnical downstream processing, J. Chromatogr. A 691 (1995) 101–112. [5] A. Kundu, K.A. Barnthouse, S.M. Cramer, Selective displacement chromatography of proteins, Biotechnol. Bioeng. 56 (1997) 119–129. [6] K.M. Sunasara, F. Xia, R.S. Gronke, S.M. Cramer, Application of hydrophobic interaction displacement chromatography for an industrial protein purification, Biotechnol. Bioeng. 82 (2003) 330–339. [7] K.A. Barnthouse, W. Trompeter, R. Jones, P. Inampudi, R. Rupp, S.M. Cramer, Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants, J. Biotechnol. 66 (1998) 125–136. ´ Sample displacement chromatography as a [8] M.S. Gajdosik, J. Clifton, D. Josic, method for purification of proteins and peptides from complex mixtures, J. Chromatogr. A 1239 (2012) 1–84.
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[17] A. Ghanem, R. Healey, F.G. Adly, Current trends in separation of plasmid DNA vaccines: a review, Anal. Chim. Acta 760 (2013) 1–15. [18] R. Freitag, S. Vogt, Preparation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography, Cytotechnology 30 (1999) 159–167. ˇ ˇ [19] U. Cernigoj, U. Vidic, M. Barut, A. Podgornik, M. Peterka, A. Strancar, A multimodal histamine ligand for chromatographic purification of plasmid DNA, J. Chromatogr. A 1281 (2013) 87–93. ˇ [20] N. Skrlj, N. Erˇculj, M. Dolinar, A versatile bacterial expression vector based on the synthetic biology plasmid pSB1, Protein Express. Purif. 64 (2009) 198–204. ˇ [21] F. Smrekar, A. Podgornik, M. Ciringer, S. Kontrec, P. Raspor, A. Strancar, M. Peterka, Preparation of pharmaceutical-grade plasmid DNA using methacrylate monolithic columns, Vaccine 28 (2010) 2039–2045. [22] A. Sousa, D. Bicho, C.T. Tomaz, F. Sousa, J.A. Queiroz, Performance of a nongrafted monolithic support for purification of supercoiled plasmid DNA, J. Chromatogr. A 1218 (2011) 1701–1706. ˇ ˇ [23] S. Cardoso, U. Cernigoj, N. Lendero Krajnc, A. Strancar, Chromatographic purification of plasmid DNA on hydrophobic methacrylate monolithic supports, Sep. Purif. Technol. 147 (2015) 139–146. [24] A. Podgornik, S. Yamamoto, M. Peterka, N. Lendero Krajnc, Fast separation of large biomolecules using short monolithic columns, J. Chromatogr. B 927 (2013) 80–89.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Sample displacement chromatography of plasmid DNA isoforms a,∗ ˇ Urh Cernigoj , Urˇska Martinuˇc a , Sara Cardoso a,b , Rok Sekirnik a , Nika Lendero Krajnc a , a,c ˇ Aleˇs Strancar a
BIA Separations d.o.o, Mirce 21, SI-5270 Ajdovˇscˇ ina, Slovenia University of São Paulo, Escola Politécnica da USP – Department of Chemical Engineering, Av. Prof. 7 Lineu Prestes, 580, Conjunto das Químicas, Bloco 18, 61548, CEP 05424-970 São Paulo, Brazil c The Centre of Excellence for Biosensors, Instrumentation and Process Control – COBIK, Velika pot 22, SI-5250 Solkan, Slovenia b
a r t i c l e
i n f o
Article history: Received 10 May 2015 Received in revised form 13 August 2015 Accepted 14 August 2015 Available online 20 August 2015 Keywords: Plasmid DNA Hydrophobic interaction chromatography Chromatographic monoliths Preparative chromatography
a b s t r a c t Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1 mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Displacement chromatography (DC) is a chromatographic technique that depends on the use of three mobile phases: a carrier, a displacer and a regenerant [1]. The carrier facilitates the load by solubilising the sample, and enables positive interaction with the stationary phase. The displacer leads displacement of the sample components from the stationary phase; during this step, individual sample components may also displace each other from binding sites on the stationary phase. Finally, the regenerant removes the displacer from the stationary phase and regenerates the column for the next chromatographic cycle [2]. DC offers several advantages over traditional chromatographic techniques, such as effective separation factors between feed solutes and high sample loading,
∗ Corresponding author. ˇ E-mail address: [email protected] (U. Cernigoj). http://dx.doi.org/10.1016/j.chroma.2015.08.035 0021-9673/© 2015 Elsevier B.V. All rights reserved.
leading to enhanced productivity [1–4]. It is usually performed in ion-exchange [5] or hydrophobic interaction chromatography (HIC) mode [6]. The development of low molecular mass displacers for protein purification in ion exchange [7] and HIC systems [6] offers the advantage of ease of separation of the displacer from purified proteins using size-based purification methods. When the displacer is itself a component of the feedstream, the method is known as sample displacement chromatography (SDC). The method takes advantage of different relative binding affinities of sample mixture components, such that during sample loading, the components compete for adsorption onto a stationary phase. SDC approach should optimally be performed under overloading conditions, since the main separation occurs already during sample loading and column capacity is thus optimally used [8]. SDC was introduced for preparative purification of peptides in reverse-phase mode [9] and has subsequently been successfully applied for purification of proteins in ion-exchange [10], affinity [11] and HIC [12] modes, using traditional porous particle chromatographic media,
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operating under diffusion mass transfer conditions. SDC approach is most useful for samples where the product binds to a chromatographic support more strongly than the impurities, because this enables fast and efficient purification of complex samples [8,11]. Due to convective mass transfer which leads to diffusionindependent chromatographic properties, the use of convective chromatographic supports, such as monoliths, is advantageous for performing DC and SDC purification of biomacromolecules over particulate chromatographic supports [10,13]. For successful SDC on particulate chromatographic supports, flow rates must be five times lower to achieve optimal sample displacement compared to standard gradient elution separation [14]. On the contrary, flow rate-independent SDC for purification of monoclonal antibodies has been demonstrated with convective media [15]. Since SD effects on monoliths are independent of column size [10], they can be used for SDC of biomacromolecules at microanalytical, analytical and preparative scales. Despite the increasing pharmaceutical potential of plasmid DNA (pDNA), which is typically purified using step gradient elution [16,17], only a single study applied DC/SDC approach to its purification [18]. The study demonstrated that conventional porous particle columns were not suited to the separation of any two substances varying considerably in molecular mass, whereas of separation of pDNA from host cell proteins (HCP) and endotoxin was achieved using a continuous bed column. Pharmaceutical-grade pDNA requires high homogeneity of supercoiled (sc) form, and chromatographic separation of sc from open coiled (oc) or linear isoforms on preparative scale is extremely challenging due to their structural similarity [19]. DC/SDC approaches have not yet been investigated for the separation of pDNA isoforms, and the main purpose of the present work was to develop a method for separation of sc pDNA from other pDNA isoforms using SDC on monolithic chromatographic supports, that would enable high resolution, high capacity and scalable pDNA purification methods in the future. 2. Materials and methods 2.1. Materials All solutions were freshly prepared using purified water which meets the requirements for European Pharmacopoeia (AQUATEHNA Biro, Zgornja Kungota, Slovenia) and analytical grade reagents. Buffer solutions were filtered through a 0.22 m PES filter (TPP, Trasadingen, Switzerland). Agarose gel was prepared from SeaKem LE Agarose (Lonza Group, Basel, Switzerland). Sodium hydroxide (NaOH), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), acetic acid, bacto tryptone, yeast extract, potassium acetate, calcium chloride (CaCl2 ), sodium chloride (NaCl), ammonium sulfate (AS), sodium dodecyl sulfate (SDS), 2-mercaptopyridine and hydrochloric acid (HCl) were from Sigma–Aldrich (St. Louis, MO, USA), EDTA was from Kemika (Zagreb, Croatia) and boric acid was from Merck (Darmstadt, Germany). 1 ,1 -carbonyldiimidazol (CDI) was purchased from Tokyo Chemical Industry (Tokyo, Japan), while histamine hydrocloride from AppliChem (Darmstadt, Germany). 2.2. Chromatographic equipment and columns All chromatographic experiments were carried out using a gradient chromatography workstations, consisting of two pumps, optionally an autosampler with various sample loop volumes and a UV detector – Smartline (Knauer, Berlin, Germany), Äkta Purifier (GE Healthcare Life Science, Uppsala, Sweden) and model HP 1200 Series chromatograph (Agilent Technologies, Santa Clara, USA). For data acquisition and control, EuroChrom 2000 software (Knauer),
Unicorn (Äkta Purifier) and ChemStation (Agilent Technologies) were used. All CIMac monolythic analytical columns (V = 0.106 mL) were provided by BIA Separations (Ajdovˇscˇ ina, Slovenia). Three different types of hydrophobic columns were used for the preparative loadings: C4 HLD, pyridine and histamine modified columns. CIM C4 HLD is a commercialised product of BIA Separations with a butyl ligand density of 1.8 mmol/mL of the support. CIM-pyridine monolith was prepared by modifying the CIMac epoxy monolith with an ethanolic solution of 2-mercaptopyridine. The achieved pyridine ligand density on the chromatographic support was 1.0 mmol/mL, determined from elemelemental analysis. Histamine was immobilised on CIMac CDI monolith leading to the neutral carbamate bond, as it is described thoroughly in reference [19]. 2.3. Cell lysis protocol and plasmid purification All experiments were conducted using two different plasmid vectors: pEGFP–N1 (4700 bp) and pMD204 (2345 bp). The pEGFP–N1 (4700 bp) was provided by DSMZ (Braunschweig, Germany). Liquid cultures were cultivated overnight at 37 ◦ C on Luria-Bertani broth containing appropriate antibiotics. Bacterial biomass from Escherichia coli strain DH5␣ containing plasmid vector pMD204 (2345 bp) was obtained from the Faculty of Chemistry and Chemical Technology (University of Ljubljana, Slovenia) [20]. Bacteria were lysed using lysis procedure according to Smrekar et al. [21]. Chromatographic purification of pDNA from clarified lysate was performed using CIMTM HiP2 Plasmid Process Pack 8 mL columns (BIA Separations, Ajdovˇscˇ ina, Slovenia) according to manufacturer’s instructions. Samples for the calibration curve and for the enzymatic restrictions were additionally treated with Amicon® Ultra centrifugal filters (10,000 MWCO) (Millipore, Cork, Ireland) to exchange buffer to 20 mM TRIS, pH 7.4 and to concentrate the pDNA concentration up to 1.2 mg/mL. Oc pDNA was prepared from the sample containing sc pDNA in 50 mM TRIS, 10 mM EDTA, pH 7.4, by exposure of the sample to 60 ◦ C for 48 h. Linear isoform was prepared enzymatically using restriction endonuclease PstI in the buffer and conditions recommended by the supplier (Thermo Ficher Scientific, Waltham, USA). 2.4. Chromatographic experiments in buffers containing high concentrations of AS All chromatographic runs were carried out on CIMac 0.106 mL columns. Analytical runs were performed in AS gradient at the flow rate of 1.0 mL/min, where the buffer A was 50 mM TRIS, 10 mM EDTA, 3.0 M AS, pH 7.4 and the buffer B 50 mM TRIS, 10 mM EDTA, pH 7.4. 500 l of pEGFP sample of different concentrations and isoform compositions was injected on the column followed by a 25 CV gradient to 100% buffer B. The selectivity (˛) between pDNA isoforms was calculated according to Eq. (1), where VR,sc and VR,oc are the elution volumes of sc and oc pDNA isoforms in mL, while V0 is the void volume of the column in mL. ˛=
(VR,sc − V0 ) (VR,oc − V0 )
(1)
For preparative chromatography, pDNA samples of different concentrations and isoform compositions were prepared in a buffer containing 50 mM TRIS, 10 mM EDTA and various concentrations of AS, pH 7.4. Frontal analyses experiments were then performed for each sample at different flow rates and pDNA loading was monitored using UV absorbance at 260 nm.
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2.5. pDNA analysis 2.5.1. Agarose electrophoresis (AGE) Chromatography fractions containing pDNA were analysed by horizontal AGE (Takara Bio, Inc, Otsu Shiga, Japan) using SYBR Safe DNA gel stain (Invitrogen, Eugene, VI, USA), 6 × Orange DNA Loading Dye (Thermo Ficher Scientific, Waltham, USA) and GeneRuler DNA Ladder Mix (Thermo Ficher Scientific, Waltham, USA). Running buffer was 1 × TBE (40 mM TRIS, 20 mM boric acid, 1 mM EDTA, pH 8.0). Gel was prepared with 0.7% agarose in 1 × TBE buffer with 1 × SYBR Safe DNA Stain. 2.5.2. Analytical chromatography HPLC analyses were conducted on an Agilent HP 1200 Series chromatograph, coupled with a multiwavelength detector. Chromatographic separations were run on a CIMacTM pDNA analytical column (BIA Separations, Ajdovˇscˇ ina, Slovenia) with bed volume of 0.318 mL. The equilibration buffer consisted in 200 mM TRIS, pH 8.0, whereas the elution buffer was 200 mM TRIS with 1.0 M NaCl, pH 8.0. The flow-rate used was 1.0 mL/min and the gradient consisted of: equilibration buffer for 1 min, linear gradient from 0% to 60% of elution buffer (0.6 M NaCl) in 1 min, 1 min hold at 60%, a linear gradient from 60% to 70% of elution buffer (0.7 M NaCl) in 10 min, and a step gradient to 100% of elution buffer. The absorbance was measured at 260 and 280 nm. Quantification of pDNA was performed using a calibration curve with pure plasmid samples of known concentrations. A serial dilution of pDNA (between 1.0 and 120 g/mL) was prepared by diluting a stock solution (1 mg/mL) with 200 mM TRIS, pH 8.0. All pDNA concentrations used for calibration curve were determined spectrophotometrically with a Smart Spec 3000 spectrophotometer (Bio-Rad, Richmond, USA). One unit of OD 260 nm in a 10 mm cuvette is assumed to correspond to 50 g/mL of a double stranded DNA. Concentration obtained by spectrophotometer was correlated with the corresponding peak area on the chromatogram obtained by CIMac pDNA analytical column. 3. Results and discussion 3.1. Sample displacement of oc pDNA by sc pDNA It has previously been demonstrated that pDNA isoforms may be separated preparatively using CIM monoliths [21] in HIC mode. The process reported used a C4 high ligand density (HLD) CIM monolith column in conjunction with loading at 3.0 M AS, washing with intermediate (1.6 M) AS to remove oc pDNA isoform and finally elution of sc pDNA with low (0.4 M) AS. Although pDNA isoforms have been successfully separated, the drawback of the described process is the high molarity of AS required, thus increasing the cost of goods for preparative purification of pDNA. This study therefore attempted to use SDC approach to decrease the AS concentration required
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during loading onto hydrophobic chromatographic supports. Optimal AS concentration range was determined by analytical separation of two pDNA isoforms in a negative AS concentration gradient (HIC conditions). Three different ligands immobilised onto CIMac monolithic support were tested: C4 HLD, pyridine and histamine. AS elution concentrations for both pDNA isoforms, column efficiency (expressed as width of each chromatographic peak = WB ) and selectivity (˛) were calculated (Table 1). Column efficiency and selectivity, rather than resolution, were parameters used for the evaluation of pDNA isoform separation efficiency, because the contribution of each parameter on SDC could be assessed separately. While all three chemistries demonstrated similar AS elution concentration for sc isoform (between 1.54 M for C4 HLD and 1.70 M for pyridine), they differed considerably in AS elution concentration for oc isoform (between 0.82 M for C4 HLD and 1.60 M for histamine). CIM-histamine demonstrated the highest efficiency and the lowest selectivity (WB for sc pDNA 1.6 min; ˛ 1.07), C4 HLD the highest selectivity and the lowest efficiency (WB for sc pDNA 6.6 min; ˛ 1.88) and CIM-pyridine demonstrated intermediate selectivity and efficiency (WB for sc pDNA 3.0 min; ˛ 1.29). Next, dynamic binding capacity (DBC) at 10% breakthrough measurements for oc as and sc pDNA isoform standards were determined in a loading buffer containing 1.75 M AS (Table 1). DBC values at constant loading conditions correlate with the strength of interaction between analyte and chromatographic surface. As expected the capacities are low for oc isoform (0.007–0.13 mg/mL) due to weaker interactions with the monolithic surface in 1.75 M AS, while the DBC values for sc pDNA differed depending on the hydrophobicity of ligand used: highest DBC (1.61 mg/mL) was observed for CIM C4 HLD (most hydrophobic matrix), whereas the lowest DBC (0.03 mg/mL) was observed for CIM-histamine matrix with lowest hydrophobicity. Another reason for larger DBC values for sc pDNA can be explained by the high supercoiled degree of sc isoform that enables the reduction of the superficial contact area, resulting in more dense packing of the plasmid onto a surface [22]. In order to prove this hypothesis, a loading of each isoform should be performed at the conditions, where binding energy for both isoforms is high enough (i.e. in 3 M AS) to achieve a chromatographic surface limited and not pDNA-monolith interaction limited DBC for each pDNA isoform [23]. We continued with the frontal analyses experiments of the mixture of pEGFP isoforms (20% oc pDNA and 80% of sc pDNA) on C4 HLD, histamine and pyridine-based supports. Three different AS concentrations in loading buffer were tested for each column. Three typical chromatograms (one for each chromatographic support) are shown in Fig. 1 while the calculated data from all 9 experiments are collected in Appendix (Table A.1) and Fig. 2. A clear double breakthrough is present in 6 of 9 experiments. AGE analysis suggested that the first breakthrough and ensuing plateau correspond to oc isoform breakthrough. The second breakthrough corresponds to sc pDNA isoform appearing together with
Table 1 Chromatographic characterisation of three different CIMac hydrophobic columns for pDNA isoforms separation. Column chemistry
cAS (oc pDNA) (M)
cAS (sc pDNA) (M)
˛
WB (oc pDNA) (min)
WB (sc pDNA) (min)
DBC50 (oc pDNA) (mg of pDNA/mL of the support)
DBC50 (sc pDNA) (mg of pDNA/mL of the support)
C4 HLD pyridine histamine
1.54 1.70 1.69
0.82 1.32 1.60
1.50 1.30 1.07
6.2 1.7 1.2
6.6 3.0 1.6
0.13 0.05 0.007
1.61 0.53 0.03
Columns 2–6: Separation of oc and sc pDNA mixture in AS descending gradient under analytical loading conditions. The AS elution concentration, selectivity (˛) and base width (WB ) of chromatographic peaks for oc and sc pEGFP-N1 isoforms on different CIM hydrophobic supports. Column volume (CV) = 0.106 mL, Q = 1.0 mL/min, = 260 nm, Vinj = 50 l, Sample: pDNA (20 g/mL) in buffer A. Buffer A: 50 mM TRIS, 10 mM EDTA, 3 M AS, pH 7.4; Buffer B: 50 mM TRIS, 10 mM EDTA, pH 7.4. Gradient from 0 to 100% buffer B in 47 CVs. Columns 7–8: DBC10 values for pure oc and pure sc pEGFP isoforms on three different chromatographic columns in a loading buffer containing 1.75 M AS. CV = 0.106 mL, Q = 0.5 mL/min, = 260 nm, Loading: sc pDNA (20 g/mL) or oc pDNA (5 g/mL) in 50 mM TRIS, 10 mM EDTA, 1.75 M AS.
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Fig. 1. Loading of a mixture of oc and sc pDNA (pEGFP) with sc pDNA homogeneity of 80% on different CIMac columns in buffers containing specific concentrations of AS. Loading buffer: 50 mM TRIS, 10 mM EDTA, different concentrations of ammonium sulfate (AS), pH 7.4. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4; Q (volumetric flow rate) = 0.5 mL/min, = 260 nm. Pyridine column: (oc pDNA) = 3.5 g/mL, (sc pDNA) = 15 g/mL, c (AS) = 2.0 M. C4 HLD column: (oc pDNA) = 4.7 g/mL, (sc pDNA) = 19.8 g/mL, c (AS) = 1.7 M. Histamine column: (oc pDNA) = 4.6 g/mL, (sc pDNA) = 19.0 g/mL, c (AS) = 2.1 M.
high concentrations of oc pDNA. Sc pDNA isoform with higher homogeneity compared to the load was detected in all elution fractions. The observation of a double breakthrough correlates well with the different DBCs for pure pDNA isoforms on different columns (Table 1). However, the appearance of a double breakthrough alone does not indicate a sample displacement (SD). SD can only be inferred by comparison of the oc pDNA concentration in the second breakthrough with its concentration in the load (Fig. 2A), where the ratio should be higher than 1. pDNA isoform concentration was quantified by CIMac pDNA analysis.
A higher concentration of oc in breakthrough compared to the load was observed for CIM-pyridine and CIM-histamine columns, suggesting that SD effect is in operation (Fig. 2A). The effect was more pronounced at higher AS concentrations: when loading concentration of AS was lowered to the proximity of the elution concentration for oc pDNA, displacement was not observed. In such case, a double breakthrough resulted mainly from the lower DBC for oc isoform. When the loading AS concentration was increased, displacement of oc molecules was observed. Effect differed for different types of hydrophobic monoliths; C4 HLD column showed a very limited SD effect regardless of the AS concentration used, likely because of a higher selectivity and broader chromatographic peaks (sc pDNA cannot displace oc pDNA because of the binding energy difference). In contrast, CIM-histamine column, where the selectivity is lowest and efficiency is highest, the SD effect was most pronounced. The results raised the possibility of extending the applicability of SDC for the removal of other nucleic acid-based impurities in pDNA downstream process, such as RNA or gDNA. Future studies should focus on extending the SDC approach to these molecules as well. 3.2. Purification efficiency of sc pDNA Since the main purpose of the work was the purification of sc from oc pDNA, it was necessary to analyse the elution fractions for all experiments as well. The sc pDNA homogeneity increased with the decrease of AS loading concentration (see Fig. 2B) for all ligand chemistries. It is speculated that a thermodynamic equilibrium between adsorbed sc and oc pDNA molecules is formed at each loading chromatographic conditions and with lowering AS concentration this equilibrium is shifted towards higher content of sc pDNA isoform. Purification of sc pDNA isoform at certain chromatographic conditions cannot exceed the thermodynamic equilibrium ratio. The correlation between the SD effect and homogeneity of the sc pDNA in the elution fraction is not straightforward. The more pronounced SD effect does not necessarily result in better homogeneity. Higher was the selectivity, less pronounced was SD effect,
Fig. 2. The detailed analysis of sample displacement effect – influence of AS concentration as well as type of the chromatographic support. A – ratio between oc pDNA concentration in breakthrough vs. its concentration in load; B – homogeneity of sc pDNA in the elution fraction. The figure is based on the evaluation of the nine different frontal analyses experiments, three of them are shown in Fig. 1 pDNA isoforms quantification in different fractions were performed using CIMac pDNA column as described in Section 2.5.2. Additional information on the experiments is included in Appendix (Table A.1).
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but an excellent homogeneity could still be obtained (for example around 98% for C4 HLD monolith). In such case, the oc pDNA was mostly removed by negative chromatography mode (the oc pDNA isoform did not bind to the matrix). On the contrary, the histamine column showed highly expressed SD effect even in 2.4 M AS loading buffer, but the sc pDNA homogeneity in the eluted fraction was just slightly improved compared to the load. At such high concentration of AS both isoforms bound much stronger to the matrix, and consequently the removal of oc form of pDNA by negative chromatography mode at such conditions became negligible and the oc isoform cannot be sufficiently removed solely by SD. Another important aspect of preparative chromatography is the amount of the purified analyte in the elution fraction. Up to 1.7 mg per mL of monolith support, the amount of eluted pDNA increased with the increase of AS concentration in the load, regardless of the chemistry used (Supporting information Table 1). Increasing the concentration of AS further would likely result in elution of pDNA at the limit of binding capacity for hydrophobic CIM monolith with average pore size diameter of 2100 nm (3 mg/mL of support) [23]. However, the homogeneity of sc pDNA in elution fractions was below 95% at the highest AS concentrations used with each specific support in this study. Therefore, when purifications are performed in SDC mode, the optimal loading AS concentration should be defined by the balance between the binding capacity and homogeneity (purity) of the final product.
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Fig. 3. Agarose electrophoresis (AGE) picture of SDC samples from mixtures of three pDNA isoforms on CIMac pyridine column. The bands from 1 to 11 on AGE gel correspond to fractions at different times during loading, presented in the inserted chromatogram. Band L is a loading fraction, while band E is an elution fraction from the column (the elution is not shown on the inserted chromatogram). Q = 0.5 mL/min, = 260 nm, (oc pDNA) = 3.0 g/mL, (lin pDNA) = 5.0 g/mL, (sc pDNA) = 13 g/mL in 50 mM TRIS, 10 mM EDTA, 1.90 M AS, pH 7.4. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4.
3.3. SDC for different plasmids In all subsequent experiments CIM-pyridine column was used at the optimal binding conditions (the amount of adsorbed plasmid and sc pDNA homogeneity) derived above. In order to demonstrate general applicability of monolithic columns for SDC of pDNA, purification was performed on a representative plasmid (pMD204, 2347 bp) (see Figure A.2 in Appendix). AS concentration in binding buffer depends on the hydrophobicity of individual pDNA molecule (influenced by base pair type composition and on the size of the pDNA [23]) and has to be determined for each pDNA sample separately. For isoform separation of pMD204 with CIM-pyridine optimal concentration of AS in loading buffer was determined at 2.0 M. The initial sample contained 30% of oc isoform; pDNA isoform quantification suggested that that 2.0 M AS led to a ratio of oc pDNA concentration in the second breakthrough vs. oc concentration in the load of 1.17, confirming displacement of oc by sc pDNA. Additionally, the homogeneity of sc isoform in elution fraction was 98%. The amount of eluted pDNA was 1.5 mg/mL of column.
Fig. 4. pDNA (pEGFP) loading and a double breakthrough under SDC conditions of samples containing different ratios between oc and sc pDNA isoform. CIMac pyridine column: (sc + oc pDNA) = 23 g/mL in 50 mM TRIS, 10 mM EDTA, 1.95 M AS buffer, pH 7.4, Q = 1.0 mL/min, = 260 nm. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4.
3.5. The effect of different oc:sc pDNA ratios in loading sample on SDC 3.4. Sample displacement of linear isoform Next, a removal of linear DNA isoform of pEGFP plasmid was analysed under SDC conditions. AS elution concentration for linear isoform was 1.68 M, similar to oc pDNA isoform (1.70 M), therefore it was concluded that SDC could work for removing linear isoform as well. A mixed sample (in 1.9 M AS) was therefore prepared containing 30% of linear isoform, 10% of oc isoform and 60% of sc isoform, and loaded on the pyridine column under SDC conditions. AGE analysis (Fig. 3) suggested that the first breakthrough correlated with the appearance of linear and oc isoforms, while the second breakthrough corresponded to sc pDNA, suggesting the enrichment of sc isoform in the elution fraction. pDNA analysis revealed a ratio of linear isoform concentration in fractions 7 and 8 vs. linear isoform concentration in load as 1.2 (Fig. 3), confirming a displacement of linear isoform by sc isoform. Additionally, the homogeneity of sc isoform in elution fraction was 98%, meaning that the enrichment from load to elution was 1.6 (Fig. 3).
In order for the pDNA purification method to be useful on preparative scale, it must apply to different oc:sc pDNA mixtures. It is known for pDNA purification using gradient elution that the AS concentration in wash buffer for oc pDNA has to be optimised for different oc:sc pDNA ratios [23]. It was speculated that SDC should be independent of the pDNA isoform ratio, because it is applied at overloading conditions. The SDC method was therefore tested on samples containing 10, 25 and 50% of oc isoform (Fig. 4). The concentration of AS in load was 1.95 M. The appearance of a double breakthrough was most evident for the sample containing 50% oc pDNA, while it was not strongly pronounced for the sample containing 10% oc pDNA. However, analysis of oc pDNA concentrations in different fractions suggested that SD operated for samples with 10 and 25% oc isoform (see Table 2), while for 50% oc isoform the concentration of oc pDNA in load and in the displaced fraction was very similar resulting in small relative difference. The homogeneity of the sc pDNA in the elution fraction
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Table 2 pDNA isoforms analysis from preparative SDC chromatography – pEGFP loading of samples containing different ratios between oc and sc pDNA isoform on CIMac pyridine column. % of oc isoform in load
Ratio between oc pDNA concentration in breakthrough fraction and in load
Amount (mg) of eluted pDNA per mL of column
sc pDNA homogeneity in elution fraction
10 25 50
1.10 1.21 1.05
1.12 1.15 1.09
97 96 95
The loading buffer contained 1.95 M AS. FT1, FT2 and FT3 fractions correspond to samples indicated on the chromatograms in Fig. 4.
4. Conclusions Sample displacement chromatography (SDC) of pDNA isoforms under HIC conditions has been demonstrated for the first time. Hydrophobic columns with different affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under overloading conditions and the separation efficiency was shown to be dependent on the selectivity of the column for different isoforms as well as on column efficiency. The SD effect often overlaps with non-binding (negative mode) effect, additionally influencing the separation efficiency. The loading AS concentration influences the purity of the elution fraction, obtaining higher purity with lower AS concentrations. The SDC was successfully implemented for the enrichment of sc pDNA for differently sized plasmid vectors, for the separation of linear isoform from sc isoform and for samples containing different oc:sc isoform ratios (even at 1:1 ratio). Like other chromatographic processes involving CIM monoliths, SD efficiency is flow rate-independent, and has been demonstrated at linear velocity of 450 cm/h. The potential of this method could be found in the preparative chromatography of pDNA because of several advantages over step gradient elution mode, such as a lower concentration of AS required during loading and removing a chromatographic step (wash) during the chromatographic process. Although DBC for pDNA is lower in SDC compared to standard pDNA purification methods, this can be avoided by utilising continuous, multicolumn chromatography systems, with which SDC is compatible, and which should be addressed in future studies. Acknowledgements
Fig. 5. SDC of pEGFP isoforms at different flow rates. CIMac pyridine column: (oc pDNA) = 3.6 g/mL, (sc pDNA) = 14.8 g/mL in 50 mM TRIS, 10 mM EDTA, 2.0 M AS buffer, pH 7.4, = 260 nm. Elution buffer: 50 mM TRIS, 10 mM EDTA, pH 7.4.
decreased from 97 to 95% with the substantial increase of the initial oc:sc ratio. However, even at 1:1 ratio of oc:sc isoforms the enrichment factor of sc isoform in the eluted pDNA fraction was very high (1.9), suggesting the ability of monoliths to purify very heterogeneous pDNA samples to pharmaceutical grade in a single step. The possibility of using the same concentration of AS in binding buffer for different oc:sc isoform ratios makes the SDC more advantageous (robust) compared to gradient elution pDNA purification. 3.6. SDC at different flow rates One of the unique features of monolith chromatographic columns is flow-rate independent separation that was demonstrated for SDC of proteins [24], but had never been shown for plasmids. Therefore, separation of pDNA isoforms (80% homogeneity) under HIC conditions was tested at 2.5 CV/min (75 cm/h), 5 CV/min (150 cm/h) and 15 CV/min (450 cm/h) flow rates (Fig. 5). The x-axis of the loading chromatograms was unified to mL in order to compare the effect of different flow rates. Elution profile, when standardised for CV/min was identical for all flow rates (Fig. 5), suggesting that SDC operates at flow rates up to 15 CV/min (450 cm/h). DBC values of 1.30 ± 0.1 mg/mL were obtained for all flow rates used, consistent with convective mass transfer of molecules within the monolithic structure [23]. Importantly, the homogenity of the sc pDNA (approx. 97.5%) in the elution fraction was not affected by the flow rate, suggesting also that shear forces are not a limiting factor when using monolithic supports at very high flow rates.
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