Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography

ABSTRACT

A novel purification method was developed for recovering the pIDKE2 plasmid, which encodes a polyprotein encompassing amino acids 1–650 of the hepatitis C virus (HCV) polyprotein, from recombinant Escherichia coli. Bacterial cells were harvested and subjected to alkaline lysis. After centrifugation, the host contaminant RNA was removed from the clarified alkaline lysate using a highly loaded size-exclusion chromatography and the eluted fraction was applied to reverse-phase media: POROS R1 50. Finally, a second size-exclusion chromatography step was carried out to purify the plasmid DNA from other small molecular-weight contaminants. Analytical methods proved that the purified plasmid DNA had a purity of 95% after Sephacryl S1000. Plasmid identity was confirmed by restriction enzyme digestion. Biological activity of the purified plasmid was confirmed in vivo; immunized mice developed a positive antibody response against all HCV structural antigens. This procedure offers an alternative to traditional methods that use organic reagents, mutagenic and toxic compounds, and animal-derived enzymes. Although the yields are lower when using this method, it is scalable and free of animal-derived substances and organic solvents.

Gene therapy and DNA immunization are becoming important alternatives for developing successful preventive and therapeutic treatments for many diseases. With thousands of people now receiving plasmid DNA (pDNA), it therefore must be produced by scalable manufacturing processes that meet stringent quality criteria in terms of purity, potency, efficacy, and safety. Plasmids are circular, double-stranded molecules that comprise approximately 1% of the total content of the host bacterial cell. They are normally isolated by an alkaline procedure, designed to disrupt the host cells and denature proteins and chromosomal DNA (chDNA), while preserving plasmid's structural integrity.¹ Although commonly used in laboratories, pDNA isolation methods that use elements such as organic reagents, mutagenic and toxic compounds, and animal-derived enzymes, are an additional concern for regulatory agencies, and therefore, must be avoided.^2,3 Another challenge in purifying plasmids is eliminating contaminant cellular components from the host, generally E. coli, which induce immunological and biological responses. RNA removal presents a challenge in producing genetic therapeutics because of the similarity in chemical composition and structure with pDNA and its high abundance in crude plasmid preparation.

Avecia

This article describes a new pDNA purification method that uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography on Sepharose CL 4B, using a buffer containing 1.5 M (NH₄)₂SO₄. The pDNA was pooled and directly applied on the POROS R1 50 media, which is a rapid alternative to conventional chromatography. For the last purification step, another size-exclusion chromatography method was chosen, because it can achieve two objectives in one operation—further purification and buffer exchange. Following these steps, the immunogenicity of the plasmid obtained was evaluated. Functionality of purified plasmids was confirmed in vivo; all immunized animals developed anti-HCV antibodies.

The final process has proved to be generally applicable and can be used from early clinical phases to market supply. Although the yields are lower, this method is scalable and free of animal-derived substances and organic solvents.

MATERIALS AND METHODS

Materials

Chemicals were purchased from Merck (Whitehouse Station, NJ) and Sigma (St. Louis, MO). Ultrafiltration equipment and membranes were provided by Sartorius (Goettingen, Germany). Design Expert Version 6.06 (DX6) software was obtained from Stat-Ease, Inc. (Minneapolis, MN).

Recombinant Proteins

Recombinant Co.120 and E1.340 were obtained from recombinant E. coli with >85% purity, by a combination of washed-pellet procedures and gel-filtration chromatography.^4,5 Co.120 comprises the first 120 aa of the HCV nucleocapsid protein. E1.340 encompasses aa 192–340 in the viral polyprotein. E2.680 comprises aa 384–680 of the HCV polyprotein and is obtained from recombinant Pichia pastoris, also by a combination of washed-pellet procedures and gel-filtration chromatography.⁶

Production of pIDKE2

E. coli DH10B cells harboring pIDKE2, a plasmid for DNA immunization expressing the first 650 aa of the HCV polyprotein from a genotype 1b-Cuban isolate, were grown over-night, at 37 °C, in 100-mL shake flasks containing 50 μg kanamycin/mL, at 250 rpm.⁷ The engineered E. coli was cultured using a complex media designed with a 50 g/L of yeast-extract concentration in fed-batch mode to produce pIDKE2 plasmid.

Lysis and Plasmid Recovery

Bacteria cell paste (typically 200 g) was suspended in 2.4 L of 61 mM glucose, 10 mM Tris-HCL, 50 mM EDTA pH 8. Lysis was performed by adding 2.4 L of 0.1 M NaOH and 1% w/v SDS, followed by incubation for 10 min at 4 °C. The lysate was neutralized with 2.4 L of 3 M potassium acetate for 10 min at 4 °C. Precipitated material including cell debris, most chDNA, and proteins were removed by centrifugation at 3,000 g for 10 min. The clarified cell lysate was concentrated five times on a tangential flow filtration (TFF) system (nominal molecular weight cutoff 100,000 kDa, 0.1 m²). All experiments were conducted maintaining constant transmembrane pressure (TMP) of 0.8 bar at 4 °C, using Sartocon Slice equipment from Sartorius. The lysate was loaded on Sepharose CL 4B in BPG 113 column to remove RNA using a 20 mM Tris-HCL, 3 mM EDTA, and 1.5 M (NH₄)₂SO₄ pH 7.2 buffer. The pDNA fraction was purified using POROS R1 50 reverse-phase media and the elution was carried out in acetonitrile at 10% v/v. The DNA polishing step was carried out on Sephacryl S-1000 media in formulation buffer: 20 mM NaH₂PO₄–Na₂HPO₄; 1 mM EDTA; and 30 mM NaCl pH 6.7 ± 0.2. Fractions containing more than 90% supercoiled plasmids were pooled and concentrated using PEG 8000 and NaCl, following methods published by Lis and Schleif.⁸

Analytical Methods

Flow-through and eluted fractions were precipitated by 1 volume of 2-propanol for 15 min on ice and centrifuged at 14,000 x g for 10 min at 4 °C. The pellet was washed with 70% ethanol and centrifuged again at 4,000 x g for 10 min. The pellet was air-dried for 5–10 min and dissolved in 50 μl of 10 mM Tris-HCl pH 8.5. Fractions prepared as previously described were tested for restriction digestion analysis by transformation experiments and analyzed by 0.8% agarose gel electrophoresis. Yield and pDNA purity were determined by measuring absorbance at 260 (A260) and 280 (A280) and expressed as A260/A280 ratio in a low-salt buffer. As a positive control, supercoiled pDNA purified by the Horn method was used.⁹

Mice Immunization and Antibody Detection

BALB/c (H-2d) female mice, 6 to 8 weeks old and weighing 18–20 g were purchased from CENPALAB (Havana, Cuba) and used for all in vivo studies. Groups of 22 animals were immunized with the mixture of Co.120 (10 μg) plus plasmid pIDKE2 (100 μg), obtained using the method described by Horn⁹ or the method described here. Mice were injected intramuscularly at 0, 3, 7, 12, and 16 weeks in the quadriceps muscle with 100-μL final volume of 0.9% NaCl-based solution. Normal saline was administered to negative control group under similar conditions. Blood samples were collected 17 weeks after primary immunization from retro-orbital sinus and the sera were analyzed for antibodies (Ab) to HCV Co.120, E1.340, and E2.680 proteins.

To detect murine anti-HCV antibodies, 96-well microtiter plates (Costar, Cambridge, MA) were coated with 100 μL of recombinant proteins Co.120, E1.340, or E2.680 (10 μg/mL) diluted in coating buffer (50 mM carbonate buffer, pH 9.6) and incubated for 12 h at 4 °C. The wells were washed three times with 0.05% v/v Tween 20 in phosphate buffered saline pH 7.5 (PBST) and blocked with 200 μl of PBST containing 1% w/v skim milk (Oxoid Ltd, England) for 1 h at 28 °C. After three washes with PBST, 100 μL of serial double dilutions of individual mouse sera in PBST were added and incubated at 37 °C for 1 h. The plates were washed three times with PBST, and 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham, UK) diluted 1: 25,000 was added at 37 °C for 1 h. Positive reactions were visualized with o-phenylenediamine (Sigma) in 0.1 M citric acid, 0.2 M NaH₂PO₄ pH 5.0, and 0.015% v/v hydrogen peroxide as substrate, and the reaction was stopped with 50 μL of 2.5 M H₂SO₄. Optical density (OD) was determined at 492 nm using a plate reader (SensIdent Scan, Merck, Ger-many).

The cut-off value to consider a positive antibody response was established as twice the mean OD 492 nm of the negative control sera.

Figure 1

RESULTS

Lysis and Plasmid Recovery

In the alkaline environment, the chDNA is denatured, while within a pH range at 12–12.3 pIDKE2 plasmid remains double stranded. During neutralization, the pH of the solution is reduced to a value close to 5.5 by adding potassium acetate. The change in physicochemical conditions of the solution causes the renaturation and flocculation of the chDNA, as well as the precipitation of protein-SDS complexes and cell-wall debris. The insoluble material can be separated from the liquid containing the pIDKE2 plasmid by centrifugation. Once the pellet is separated, the clarified alkaline lysate containing the pIDKE2 plasmid is concentrated 5-fold using a TFF system. This step, however, is not enough to remove all the RNA (Figure 1, Lane 4). On the other hand, the 260 nm/280 nm absorbance ratio was 1.8, which is in the range of the purity parameter regulated for a drug substance containing a purified pDNA.¹⁰ RNA removal can be performed enzymatically using RNase or by selective precipitation using Ca2+; NH4+, or Mg2+ ions or polyethylene glycol.^9,11 Depending on the qualitative and quantitative composition of the sample, this polymer involves the risk of co-precipitation of DNA and needs to be removed before subsequent ion-exchange steps. RNA removal also can be achieved by size-exclusion chromatography using Sepharose 6 Fast Flow, which recovers 92% of the pDNA. In this process, RNA was removed from the concentrated cell lysate during the first chromatographic step on Sepharose CL-4B recovering 91% of the pDNA by group separation in the presence of buffer containing 1.5 M (NH₄)₂SO₄.¹² The use of high salt concentration is beneficial because of the different hydrodynamic size of doubled-stranded pDNA compared with small single-strand content such as RNA and other nucleic acids impurities. Therefore, pDNA eluted in the void volume can be clearly separated from RNA (Figure 1, Lane 5). The elution profile, obtained during the pDNA elution from Sepharose CL-4B in the presence of (NH₄)₂SO₄, is shown in Figure 2.

Figure 2

The second step was designed as a concentration step, and therefore, a support with high capacity for large molecules was used: a reverse phase POROS R1 50 matrix with a dynamic binding capacity between 5 and 1.5 mg pDNA/mL using a high flow rate (500 cm/h). The high flow rate is the main advantage of POROS. It does not affect the resolution or capacity during pDNA purification because of the intraparticle, convective, solute transport, which is a fundamental new approach to reducing mass-transfer limitations in chromatography. This new approach will dramatically decrease separation time and increase throughput and productivity for pDNA recovery.¹³

Figure 3

Another size-exclusion chromatography method was chosen as the last purification step. Agarose gel electrophoresis of column fractions showed that purity of the pDNA increased from the first fraction, which had <60%, to the last, in which purity of >95% was obtained (Figure 3). The pDNA fractions with a purity >89% were pooled and further concentrated by a 10% w/v PEG-8000 precipitation, which concentrated the plasmid and reduced levels of E. coli host proteins and undesirable DNA contamination.⁹ The pellet was resuspended in the formulation buffer at 2 mg/mL and was finally filtered (0.22 μm) with a yield of about 100% before further analyses were performed. Analytical methods were followed according to the criteria recommended by the FDA. A summary of the analytical specifications and final results for pIDKE2 is shown in Table 1.

Table 1. The purified plasmid DNA had 95% purity. Contaminants such as host-cell RNA and DNA were undetectable by agarose gel electro-phoresis assay. Plasmid identity was confirmed by restriction enzyme digestion and activity was confirmed by an in vivo assay, in which the results show that the purified pIDKE2 plasmid induced a positive and long-term antibody response against HCV core protein and enveloped proteins.

Immunogenicity Study

The immunogenicity of pIDKE2 plasmid obtained by the method described in the current work was compared with another method previously described.⁹ All animals immunized with pIDKE2+Co.120 developed anti-HCV antibodies. Figure 4 shows the antibody response against HCV structural antigens at week 17. Mean antibody titers above 1:800 were elicited against HCV structural antigens in animals immunized with the mixture of pIDKE2 and Co.120. In fact, antibody titers against Co.120 and E2.680 reached values above 1:2,000. No significant differences were observed in antibody titers generated by immunization with the plas-mid obtained by different procedures. Mice immunized with normal saline did not induce any detectable antibody response.

Figure 4

DISCUSSION

Historically, highly purified pDNA recovery has been accomplished through the use of cesium chloride/ethidium bromide (CsCL/EtBr) buoyant density gradient separation.¹⁴ This method allows the separation of pDNA by buoyant density into purified bands of different forms: supercoiled (sc), open circular (oc), linear (l) and multimeric (m) plasmid. Although it yields a highly purified plasmid, this approach is not scalable because of personnel safety issues and the hazardous waste considerations associated with the use of cesium chloride and ethidium bromide. Using these process solutions at large scales requires safety measures such as designing explosion-proof facilities or using appropriate protection. In addition, the use of ultracentrifugation is also a major impediment to the scale-up of this technology. In contrast, simple unit operations and the avoidance of critical reagents such as animal-derived compounds (e.g., enzymes), detergents, and organic solvents significantly reduce the need for validation efforts and precautions to ensure patient and operator safety.

Our pDNA purification process is based on alkaline lysis, TFF, and size-exclusion chromatography as primary downstream steps for extensive removal of RNA. Reverse phase interaction chromatography is then used to purify the pDNA from the remaining impurities, particularly because of its ability to reduce the endotoxin burden to levels below the specifications. Volume reduction of the resulting stream is achieved by precipitation of the plasmid with PEG instead of 2-propanol or ethanol. Finally, size-exclusion chromatography is used as a polishing step and to exchange the buffer for an adequate formulation. The proposed process does not use or generate significant amounts of hazardous materials and no special safety requirements are envisaged. Thus, environmental or safety-associated costs are minimized. The reagents used do not pose any special regulatory concern because they are nontoxic, nonmutagenic, and nonflammable.

It is also strongly recommended to spend sufficient time and efforts developing large-scale GMP processes. This may result in a different approach when compared with 'kit' protocols, in which convenience and simple robustness play the most important role. Depending on the final application as a therapeutic (high-dose single injection, or long-term, low-dose treatment) or for diagnostics, specific demands may require individual solutions. Given the complexity of the starting material, certainly single purification step will not be enough to meet the regulatory requirements. Nevertheless, the aim is to establish a robust and preferably generic protocol that is applicable to a variety of plasmids of different sizes (regardless of individual precautions related to stability or sensitivity to shear forces). When developing a multistep large-scale pDNA purification process, the design will aim to begin with fast volume reduction. This can be achieved by ultrafiltration or any (chromatographic) capture step, in which recovery (>90%) is more important than maximal capacity.¹⁵

Currently published processes for pDNA purification include precipitation and extraction of pDNA by organic solvents, ultrafiltration, and predominantly liquid chromatographic techniques. Most of the available processes for pDNA purification are time-consuming and not scalable. Furthermore, because these processes use materials that are not certified for application in humans and also enzymes of avian or bovine origin, these processes do not meet the appropriate regulatory guidelines.

Chromatography is considered the highest resolution method, and therefore, it is essential for producing pDNA suited for therapeutic applications. The most commonly used techniques for initial purification are anion-exchange and hydrophobic interaction.^2,16 It has to be considered that the large pDNA molecules adsorb only at the outer surface of particulate supports.¹² Consequently, capacities are usually on the order of hundreds of micrograms of plasmid per milliliter of chromatographic support. In our process, we used a POROS R1 50 reverse-phase matrix, which has a dynamic binding capacity between 5 and 1.5 mg pDNA per mL support. Finally, as a polishing step, size-exclusion chromatography is the most suitable for removing undesired pDNA isoforms and host cell proteins and to achieve buffer exchange. The processes recover 95% of pDNA similar to the process described by Horn, et al.⁹ The results demonstrate that this process meets all regulatory requirements and delivers pharmaceutical grade pDNA. The final chDNA content is <5 μg per dose, RNA is not detectable by agarose gel electrophoresis, protein content is lower than 5 μg per dose, and the endotoxin content is 0.6 EU per kg of body weight.

CONCLUSIONS

The clinical application of gene therapy and DNA immunization will depend not only on efficacy but also on safety and the ease with which the technology may be adapted for large-scale pharmaceutical production. The new purification process described here combines novel and conventional purification technologies as high-throughput alternatives. TFF membranes and a perfusion media were used for concentration and recovery of the pIDKE2 plasmid to reduce process time and to increase the productivity of the downstream process. Although the yields are lower when using this process, the advantages of this method over existing plasmid DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. Finally, this process may be used as a simple, scalable, and applicable method for the production of the pIDKE2 plasmid, which is being used in Phase 2 human clinical trials.

Miladys Limonta is the principal researcher, Gabriel Marquez is the head, and Isabel Rey is a technician in the downstream process development department, Martha Pupo is a specialist and Odalys Ruiz is a researcher in the analytical development department, Yalena Amador-Canizares is a researcher and Santiago Duenas-Carrera is the head of the hepatitis C vaccine department, all at the Centre for Genetic Engineering and Biotechnology, Havana, Cuba, +53.7336.008, miladys.limonta@cigb.edu.cu

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