USES OF PLASMIDS:
Large numbers of purified plasmid (pDNA) material are required for cloning, DNA vaccine and Gene Therapy applications. Amounts required will vary with the ultimate application, and this will vary from ug scales for laboratory work up to potentially gram scale for large scale therapeutic dose applications.
PURITY REQUIREMENTS:
Purity requirements may vary, but for most applications it is critical to remove host cell products such as proteins, genomicDNA (gDNA), RNA, endotoxins and degradation products of the pDNA such as open circular (OC) and in some cases linear plasmid material. For human therapeutic use, such impurities must be removed to specified limits laid down in regulatory guidelines.
Every proposed use must define a specification for purity and in some cases potency. It is traditionally important to isolate and concentrate therapeutic materials such as pDNA to the highest possible titre, but as concentration is increased, there is a consequential risk of enhanced loss of yield due to aggregation and degradation due to shear forces and enzymic activity or sensitivity to adverse environmental conditions such as salt concentration or pH etc. Final stages of purification include steps to control product stability and to enhance shelf life and potency of the final product.
CURRENT PRODUCTION TECHNOLOGY:
Large scale production of plasmid material typically involves genetic modification of E.coli cultures and large scale fermentation production followed by harvesting the pDNA produced within the host organism.
One major difference exists here in comparison to other protein based biological therapeutics such as antibodies and recombinant protein products. Such protein products are often in significant excess and are a major fraction of the end fermentation mass, whereas plasmids are normally in very low concentration. This poses a practical problem for the isolation and purification downstream, in terms of product contamination and restricted column capacity.
There has developed an industrial standard approach to downstream recovery of pDNA from such upstream production methods. This includes a series of familiar technologies including precipitation, centrifugation, filtration in various formats including TFF and also chromatography. The most selective of these is without doubt the chromatography, and three routine modes are frequently used, HIC, IEC and SEC. Each of these poses its own problems and offers its own advantages:
· IEC is a high capacity mode which can offer major selectivity for bioproducts when managed effectively. Q-type ion exchange resins are particularly prone to irreversible fouling by DNA species and are not cleanable and therefore not reusable.
· HIC offers a practical complementary separation mechanism to work in partnership with IEC to provide extra selectivity, but uses large amounts of Ammonium Sulphate which creates a disposal issue. HIC is also an inherently lower capacity technology than IEC and a capture step based on HIC therefore demands larger columns and uses larger quantities of buffers as well as being inconvenient for handling and disposal.
· SEC is inherently a low capacity technology, and is best reserved for final stage polishing for this reason. As a non- adsorption technique it dilutes material, uses large columns and requires material concentration normally by TFF prior to use.
· TFF as a technique is not a low shear technology and typically its use involves significant loss of yield.
DISADVANTAGES OF TRADITIONAL METHODS:
All TRADITIONAL particulate chromatography methods typically rely on diffusive mass transport which limits the flow rates and mass throughput for bioproduct purification, especially those with larger species such as pDNA. The slow run times are perhaps the reason why chromatography is considered a problem method.
Traditional methods mostly use a HIC capture step, which is both a lower capacity method and involves the use of large volumes of concentrated Ammonium Sulphate (3 volumes of 4M Am Sulphate to 1 volume of lysate. This causes a major waste removal issue, together with handling inconvenience. This salt is messy to work with and poses a significant cleaning problem.
Alternative use of SEC as an initial step involves large columns which must run slowly to remove protein contaminants by size exclusion. All large species are recovered in a single elution, with no removal of endotoxins and gDNA. In addition. The plasmid material requires TFF concentration prior to loading, which promotes shear forces and loss of yield. This additional step also takes extra time.
THE MONOLITH ADVANTAGES:
FAST PURIFICATION and HIGH PURITY deliver DRAMATICALLY HIGHER PRODUCTIVITY...
Monolith chromatography materials remove the essential link between flow rate and mass transfer performance, by utilising convective flow rather than diffusive flow for mass transport for adsorption and desorption. In short, adsorbates such as products and impurities are all pumped through the large throughpores. This means that chromatography can be run at much elevated volumetric flow rates compared with traditional particulate packed columns. Typical Column volume flowthrough rates are measured in 20 or more CV per MINUTE compared with hours for particulate resin columns...even with large species such as plasmids.
DIRECTLY SCALEABLE PLATFORM METHOD... SHOW A TABLE TO COMPARE STEPS AND TIMES...
The monolith method can be used with no modification to produce any amount of purified plasmid product at scales between 6mg or less and up to 48 g per step operation! All scales can be run to cGMP compliance with full regulatory support documentation. The method is qualified to work effectively with plasmids up to 20kbp, and has been shown to work for materials of 110kbp.
MONOLITH OPERATION is a LOW SHEAR TECHNOLOGY...
Traditional packed resins create turbulent flow around particles within a chromatographic bed, which results in shear forces which can damage intact plasmid material. Monolith columns avoid such turbulence and effectively provide laminar flow which promotes a low shear environment even at elevated flow rates. This results in enhanced yield of active SC pDNA material with lower degradation.
THE METHOD ADVANTAGES:
Monolith chemistries typically involve higher ligand densities compared with traditional particulate resins. This provides a major advantage which can be exploited for plasmid purification. By using a “weak” anion exchange resin at a high salt load condition, IMPURITY PROTEINS FLOW THROUGH DURING THE CAPTURE STEP, thus maximising capacity for the desired target plasmid and minimising protein binding . SMALLER COLUMN VOLUMES are required for selective capture. In conjunction with REDUCED BUFFER VOLUMES this translates into SIGNIFICANT COST SAVINGS compared with traditional chromatography resins.
1 The use of DEAE ion exchange for plasmid capture means that the column can be cleaned and reused if necessary, since it is not fouled with gDNA as would be a Q strong ion exchanger. The columns have a longer life time in use, and the chemistry also TOLERATES EXPOSURE TO 1M NaOH for CIP, and even 2M for short exposure times (~1hr).
2 The method is RNase free, which means it is better suited to manufacturing scale since there is no need for RNase removal validation.
3 RNA is precipitated by 0.5M CaCl2 as a simple additional 15 minute step following normal alkaline lysate preparation, prior to normal centrifugation and filtration in preparation for chromatography. Residual small amounts of RNA are eluted from the DEAE capture column in a simple step elution prior to pDNA recovery.
4 Genomic DNA and endotoxins are more tightly bound than pDNA and cleared after recovery of pDNA in a simple cleaning step if necessary prior to column reutilisation.
5 A single DEAE capture and elution delivers plasmid of around 95% purity. Up to 6mg can be purified in a single step from standard lysate preparations.
6 If it is necessary to purify SC plasmid material to higher purity (~99%), a second monolith HIC purification step will deliver up to 3mg per ml of monolith column. The 1ml column will thus process 6mg from the 1ml DEAE column in just two to three passes with an identical geometry column. The HIC column is designed to remove closed circle (CC) pDNA forms and to yield high quality purified plasmid suitable for most applications.
7 Monolith chemistry allows reversal of the steps of HIC and IEC, and the high capacity step is placed in front of the lower capacity step which offers advantages of scale containment, and the volumes of Ammonium Sulfate are also reduced. The volume of HIC buffers saved is typically between x 10 less salt compared with traditional methods and even x 3 less compared with combined particulate/monolith methods.
MAJOR ADDITIONAL ADVANTAGES:
· The MONOLITH pDNA platform METHOD is available, if required, to cGMP regulatory compliance.
· It is not a patented method and is fully available to all as a Licence-free method for full scale manufacturing.
· The method can be run on a column volume basis for loading elution and cleaning, which removes the need for sophisticated and costly gradient chromatography systems and rigs which require detectors and fraction collectors etc. The columns can be run if required with low pressure pumping systems such as a peristaltic pump, running below 3 Bar. Whilst flow rate will be reduced, and run times may be extended, large volumes can be processed within a few hours with no compromise to product quality.
· If necessary however, columns can be run at elevated flow rates in the region of 20 CV per MINUTE, to deliver a FAST and EFFECTIVE method for PURE PLASMID PREPARATION if necessary at cGMP MANUFACTURING SCALE.
FINAL FORMULATION:
Material recovered from the HIC column can be buffer exchanged by SEC or TFF, but it can be quickly reloaded onto a DEAE column and recovered at high concentration with a NaCl elution followed by dilution or precipitation with isopropanol.
KEY FEATURES OF THE PROCESS:
1. RNase free method
2. CaCl2 removal of RNA and elegant concurrent removal of HCP material with IEC selective capture
3. High capacity DEAE IEC step for capture and bulk volume reduction with low buffer consumption and smaller columns
4. Reusable, cleanable DEAE IEC chemistry
5. Massive reduction in Ammonium Sulphate and reduced waste handling costs
6. Simple, predictable scale up method
7. Simple two step to purity
8. Matched column volumes for convenient system hardware configuration
9. Operation at low pressure or elevated flow with no compromise to product quality
10. Stepwise elution method operation
11. cGMP compliance if required
12. No licence fees for the process