Alkaline lysis for pDNA using a static inline mixer

Controlled alkaline lysis of E. coli cells using single-use static inline mixer to release plasmid DNA (pDNA) for further purification and use in biopharmaceutical manufacturing

Plasmid DNA (pDNA) is a prerequisite in the growing biopharmaceutical field of mRNA and in gene therapy manufacturing. Plasmids used in BioProcess™ applications are commonly in the range of 5 to 20 kilobase pairs (kbp). Here we present a controlled scaled-up process using single-use equipment and consumables for an E. coli cell line with 6.3 kbp model plasmid.

Individual lysis process parameters can be adjusted for optimization with the goal of maintaining pDNA quality and yield. We also show how the lysis and clarification process fit into the pDNA workflow and can be used in scaled-up pDNA processes from 50 to 200 L.

Previously published material around pDNA production purification and analysis from Cytiva is available in the following application notes:

1. A scalable single-use two-step pDNA purification process
2. Three-step supercoiled plasmid DNA purification

Introduction

pDNA is an important genetic engineering tool used to clone and amplify or express genes for biotechnology applications. pDNA of good manufacturing practice (GMP) grade has many applications including DNA vaccines and gene therapy, with the production of viral vectors and mRNA being dependent on the production of pDNA (1).

With the increasing use of plasmid vectors in different therapeutic areas, the focus on manufacturability, as well as regulatory requirements for these processes, have increased. Manufacturers of pDNA must comply with GMP principles even when plasmids are not themselves therapeutic (Fig 1 below).

Fig 1. Overview of where GMP principles apply for pDNA manufacturing. Adapted from reference 2.

Plasmids are amplified through a microbial system in a fermentation process followed by a series of purification steps to reach the purity and quality goals. A critical step in the purification process is alkaline lysis, where concentrated cells are suspended at neutral pH and lysed by adding an alkaline solution containing detergent. The lysate is then neutralized, resulting in the immediate formation of a white precipitate or flocculate consisting mainly of cell debris, denatured proteins, and genomic DNA.

The lysis is often performed in batch mode and removal of the flocculate/precipitate is often a centrifugation operation at lab scale. However, the batch mode of the lysis process is difficult to close and scale when fermentation volumes increase.

An additional challenge when performing lysis in batch mode is achieving optimized mixing while minimizing shear during the brief lysis period, which typically lasts only a few minutes at alkaline pH. Prolonged lysis time risks the degradation and denaturation of supercoiled plasmids (SC), while high shear forces risk fragmenting genomic DNA. Both issues can complicate the purification process and reduce yield and quality.

While batch lysis using mixers is available and performed at small scale, control of the lysis process while maintaining appropriate mixing and lysis time parameters can be challenging as cell mass and volumes increase.

Our main objective of this work is to incorporate a controlled and scalable process for E. coli lysis with single-use equipment and consumables. The pDNA workflow typically includes the lysis step between the cell concentration and clarification depth filtration process steps in the midstream process (Fig 2).

Fig 2. An overview of pDNA production through the midstream process. Lysis was performed directly from diluted cell suspension feed using the single-use static inline mixer.

The alkaline lysis process with the static inline mixer allows for optimized mixing and reduced shear to avoid fragmentation of genomic DNA and denaturation and degradation of SC plasmid. The single-use components allow easy setup, reduce the need for post-use cleaning, and ensure a closed system for the cell concentration and lysis process.

This application note shows the performance of a static inline mixer for alkaline cell lysis and pDNA release from cell suspension feed material. We performed the work at the 50 and 200 L fermenter scale using a single-use centrifuge for cell concentration, followed by a full pDNA midstream process including alkaline lysis operation, calcium chloride precipitation of RNA, clarification through flocculate lift (2), and depth filtration.

Materials and methods are described in detail at the end of the document.

Results and discussion

We produced the pDNA plasmid starting material for two runs in Xcellerex™ XDR-50 MO and XDR-200 dual-purpose (DP) fermenters. E. coli cells were concentrated, washed, and buffer exchanged using a continuous single-use centrifuge where the plasmid-containing cells were retained (heavy phase). The fermenter medium (light phase) was set to waste.

The cells in the heavy phase were retained in a circulation tank, recirculated in the centrifuge, and washed with cell suspension buffer (50 mM Tris, 50 mM glucose, 10 mM EDTA, pH 7.5), while keeping the volume constant, equivalent of > two buffer exchanges.

We determined the wet cell weight (WCW) of the E. coli cells and diluted to a final concentration of 15 L/kg of cell suspension buffer to WCW. For the 50 and 200 L fermenter runs, the recirculation vessel and dilution vessels were single-use Allegro™ 50 L and Xcellerex™ XDUO-200 single-use mixers, respectively. The starting weights of the lysis material were 50.7 and 100.0 kg with processed wet cell weights of 3.44 and 6.66 kg for the two experiments (for the 200 L run only 50% of the WCW from the cell concentration step was further processed through the lysis).

The lysis process employed single-use components throughout, including the static inline mixer assembly, mixer bags, and ReadyToProcess™ tubing assemblies. The ½” static inline mixer is a 19 cm long, 12.7 mm o.d., helical mixer. The single-use components, peristaltic pumps, and pressure monitor were organized on a ReadyKart™ mobile processing station.

The cell suspension feed material was mixed at 30 rpm in the Allegro™ 50 L mixer through the process until reaching the minimum 2 L agitation volume to keep it heterogeneous through the lysis process. The static inline mixer flow rate was controlled at a total flow rate of 500 mL/min through the mixer (per element if multiple elements are used Fig 3B). Half of the flow came from the cell suspension and half from the lysis buffer (0.2 M NaOH, 1% SDS) giving 1:1 proportion of the two solutions.

The 500 mL/min flow rate corresponds to a flow velocity of 0.067 m/s. The target was 1 min of lysis time and the length of the 1/2” i.d. tubing, post static inline mixer was scaled to match this time (4 m for the 50 L run and 7.8 m for the 200 L run). Tubing was coiled in ~ 0.3 m (1 ft) diameter loops before the introduction of the neutralization buffer (3 M potassium acetate, 2.07 M acetic acid at 2°C to 8°C) at a Y junction followed by collection into single-use mixers (XDUO 200 L mixer for the 50 L run and XDUO 500 L mixer for the 200 L run).

Figure 3 shows the setups of the runs.

Fig 3. Setup for the pDNA lysis process for the 50 L fermenter scale (A) and 200 L fermenter scale (B) using two static inline mixer elements to optimize process time. (C) Post-lysis operations with calcium chloride and ammonium bicarbonate.

The inlet pressure of the static inline mixer was consistent through the entire runs at 0.25 to 0.35 bar (Fig 4) and showed no sign of clogging during the runs. Processing time for each of the runs was ~ 3 h.

Fig 4. Pressure before static inline mixer(s) during lysis runs.

The final neutralized lysate weighed 147.7 and 311.1 kg, collected into XDUO-200 or XDUO-500 mixers for the 50 and 200 L runs, respectively. The lysis process yielded > 90% of pDNA released from the lysis process based on pDNA titers in the neutralized lysate product as compared to titer from fermenter samples processed with pDNA miniprep kits (Table 1).

Product quality data (Table 2) post-lysis shows that the static inline mixer lysis did not affect the pDNA supercoiled/open circular (SC/OC) ratio and product impurities between runs.

Table 1. End of fermentation through lysis process data. End of fermentation concentration performed via pDNA miniprep kits and spectrophotometry. Continuous centrifugation process step yield was calculated based on the percentage of cell mass at the end of fermentation compared to the continuous centrifugation amount¹

¹End of fermentation sample pDNA concentration analysis was performed using a NanoDrop microvolume spectrophotometer on batch lysis material purified using DNA miniprep kit (see Materials and methods for additional information).
²Continuous centrifugation pDNA amount data was extrapolated based on process step yield and WCW retained during continuous centrifugation.
³The 200 L run only forward processed 50% of the material from the cell concentration through the lysis process step. The step yield and overall process yield data were calculated accordingly. The neutralized lysate sample pDNA concentration analysis was performed via the Sepharose™ HP chromatography resin method (3) (see Materials and methods for additional information). The 200 L Xcellerex™ XDR fermenter run had a paired yield excursion with continuous centrifugation and neutralized lysate step yields. The overall process yields at the neutralized lysate stage were comparable between the 50 and 200 L runs.

Table 2. Fermenter and lysis analytical data¹

¹See Materials and methods section for additional details on analytical methods.

We recommended that you perform lysis optimization for specific E. coli cell lines and plasmids to improve product quality and yield. Some key parameters for the lysis process to optimize are:

• Cell suspension dilution
• Lysis and neutralization buffer composition and detergent concentration
• Lysis time/flow rate

Post-lysis, a calcium chloride precipitation, and flocculation lift with ammonium bicarbonate was performed within the Xcellerex™ XDUO mixers. We added 5 M of calcium chloride buffer to precipitate and reduce RNA. We also added 120 g/L of ammonium bicarbonate buffer to bring the precipitated material (precipitated RNA and cell debris) to the top of the mixer bag and separate it from the lysis suspension with the target pDNA. This allows the lysis material to be depth-filtered without loading the precipitated material onto the depth filters, which increases the filtration load capacity. Additions of both buffers occurred through the bottom port for addition close the mixer (Fig 3C).

We performed depth filtration with Stax™ disposable depth filter capsules and PDP8 filter membranes from the bottom outlet of the Xcellerex™ XDUO mixer. Filtration was stopped when the filtration of precipitated material started. Cake filtration with the K900P depth filter and filter aids (Celpure C300 and Harborlite 900S) was tested on the precipitated lysis material at small scale with similar results (data not shown) to flocculate lift material shown in this study for additional clarification options.

Step yields and individual analytics for the precipitation through depth filtration process steps were not quantitated until after tangential flow filtration (TFF) 1 buffer exchange due to chemical incompatibilities with assays.

Conclusions

• Single-use, static inline mixer for E. coli lysis and pDNA release provides a controlled and scalable lysis process with step yields around 90%.
• Processing of a 50 L fermenter batch with one static inline, single-use mixer in ~ 3 h.
• Process steps can be closed operations without the need for cleaning in place (CIP) of equipment.
• Ability to optimize lysis parameters for product quality and process yield targets.
• Multiple single-use mixer sizes available for use (½” and ¼” i.d. static inline mixers) in tubing sets.
• Scalable clarification process (precipitation or flocculation lift and depth filtration) for efficient removal of cell debris and precipitated material after lysis.

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