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Process and plasmid design optimization, disposable equipment, and flexible platform processes all play important roles.
Demand for plasmids is rising dramatically as large numbers of messenger RNA (mRNA) vaccines and viral-vector-based therapeutics advance through clinical trials toward approval and commercialization. The upstream manufacturing process is an important part of bringing plasmid DNA (pDNA) materials to market, as achieving the highest yields possible at large scale is essential to meeting future demand. Fermentation is anticipated to be the predominant method for large-scale production of high-quality pDNA products.
For conventional biologics produced via fermentation, upstream operations have been associated with fermentation and harvesting. In the context of plasmid bioproduction, however, it is also common to include cell lysis and clarification as part of the upstream process, according to Nuria Gomez Santos, head of process and analytical development for pDNA with Catalent Cell & Gene Therapy. “The cut off is established based on the presence (upstream) and absence (downstream) of bacterial cells or cell debris in these process unit operations,” she explains. Catalent applies such a cut off using a 0.2-micrometer filtration step to further segregate upstream and downstream operations.
Some companies, says Karl Varadi, lead process development manager for pDNA and mRNA at AGC Biologics, regard resuspension of harvested biomass, subsequent alkaline lysis, clarification, and concentration/diafiltration as midstream processing (MSP), with capture, polishing, with other related purification, formulation, and fill/finish steps are part of downstream processing. Given the importance of lysis, neutralization, and clarification in pDNA production, John Bowen, senior director for Nucleic Acids and Plasmid Operations at The Center for Breakthrough Medicines, agrees that these steps should be thought of separately, as mid-stream unit operations.
For the purposes of this article, however, lysis and related activities will be included in the discussion of upstream processing for pDNA manufacturing.
The expectation that drug manufacturers take a risk-based approach to production has drawn attention to the manufacturing practices employed for key materials used in the production of drug products, even if they are not intended to be present in the final drug product. Such ancillary materials include plasmid DNA used as a template for the production of mRNA and for the production of viral vectors used for delivery of genetic cargo. In general, the industry is moving towards the use of the highest-quality materials for both clinical and commercial manufacturing of not only critical raw materials, but ancillary materials as well. Most pDNA manufacturers consequently offer three different product grades for phase-appropriate use: research, intermediate, and current good manufacturing practices (CGMP).
“The different quality grades are aimed at different applications,” states Martin Schleef, CEO of PlasmidFactory. “For initially testing a new therapeutic gene candidate or gene delivery strategy, a research-grade plasmid would be sufficient. Further refinement of the research process results in the intermediate and highest, GMP-grades for clinical and commercial applications,” he observes.
Plasmids for use in mRNA and viral-vector manufacturing may be intermediate- or GMP-grade, depending on the preference of the drug manufacturer. Plasmids used in direct clinical applications, such as for DNA vaccines, must be of GMP-grade, according to Marco Schmeer, project manager at PlasmidFactory.
“The higher quality grades,” Schleef says, “generally are produced in dedicated manufacturing spaces, undergo more extensive quality-control testing as part of an active quality management system, and have greater documentation packages.”
The quality grade does not have significant impact on upstream and downstream process operations, however, according to Santos, as the same platform process is executed to generate R&D, GMP-like, and GMP-grade plasmids. Indeed, there should be no impact on process development if the upstream process is created with manufacturing considerations in mind from the outset, Varadi agrees. “A well-defined upstream process is ideally initiated and designed via down-scaling of an existing GMP manufacturing infrastructure, in order to compensate for technical differences. This approach allows for the generation of a robust and easily scalable process, which then just needs completion by GMP/GMP-like documentation,” he comments.
At Catalent, for instance, R&D-grade material is generated using a downscale model operated with the same parameter set points as in its cleanrooms, Santos notes. This representative R&D material has been used for process development purposes and pre-clinical studies. Both GMP-like and GMP-grade plasmids are produced using fully traced raw materials and fully disposable qualified equipment. Operations are identical as only the shared documentation and level of quality oversight varies between these grades.
The increasing amount of pDNA required for therapeutic and vaccine applications has drawn plasmid design and fermentation strategies into the spotlight. To help prepare for the expected significant market demand, the increasing amount of required pDNA for therapeutic and vaccine applications has drawn plasmid design and fermentation strategy into the spotlight, Varadi contends.
Plasmids are typically produced recombinantly in E. coli strains through a high biomass fed-batch fermentation process. High titers can be achieved through multiple means by deregulating the repression on plasmid replication, according to Matthias Craig, senior manager for Product Development at Catalent Cell & Gene Therapy. “High yielding plasmid fermentation processes have been developed and fine-tuned over the past two decades,” he states.
Implementation of fed-batch protocols has led to improved pDNA yields and increased batch success rates and optimized cost efficiency, Varadi agrees. He adds that understanding the molecular mechanisms that modulate the plasmid copy number and implementing them in next-generation plasmid backbones will help further boost yields for plasmids with more problematic genetic payloads.
“Fermentation has indeed become more sophisticated to achieve the desired goals in plasmid production such as plasmid yield per biomass,” Schleef observes. He cautions, though, that more complex issues like maintaining plasmid structural stability still remain to be addressed.
Some of the biggest factors for upstream pDNA process development include media optimization, fed-batch strategy, and Escherichia coli (E. coli) strain selection, Bowen comments. Engineering of improved E. coli host strains has, according to Craig, to some extent become more of a focus recently than fermentation process optimization. “These strains are characterized by higher fermentation titers and/or lead to increased stability of nucleic acid sequences that are prone to deletion/recombination events, such as inverted terminal repeats (ITRs) and long polyA tails,” he explains.
Another important advance noted by Craig is the development of both batch and continuous cell lysis approaches that afford high recoveries and accommodate a wide range of production scales. The adoption of single-use equipment in upstream operations has, meanwhile, significantly reduced the probability of cross-contamination, which is very important in a multiproduct facility producing a multitude of plasmids, Bowen observes.
There are many challenges that manufacturers must navigate when implementing upstream processes for pDNA production. First and foremost, very high volumetric yields are achievable for pDNA, but such yields are much lower than those achievable for overexpressed, non-toxic recombinant proteins, says Ram Shankar, manager of R&D for PlasmidFactory.
“That being said, simply overproduction of plasmid per cell would not go very far if plasmid stability and homogeneity cannot be guaranteed. Such aspects have to be considered when designing an upstream process for plasmid production,” Shankar notes. “In this context,” he continues, “we have developed strategies that keep productivity high without having to rely on antibiotics while controlling for the maintenance of critical sequence structures in the product (e.g., ITR sequences for productive adeno-associated virus [AAV] manufacturing or polyA tracks for mRNA manufacturing).”
Another common issue, according to Varadi, is achieving high-yield manufacturing of plasmids that contain difficult genetic payloads (gene-of-interest size, base composition, toxicity, use of selection markers). Larger plasmids that impose a significant metabolic stress on the host cell, meanwhile, can reduce culture fitness, leading to bad growth and low plasmid copy number. He adds, though, that narrow control of carbon and nitrogen source feeding and use of alternative non-antibiotic selection markers can reduce metabolic burden on the host cell.
It is recommended, Craig emphasizes, that scientists and drug developers verify that their plasmid constructs are of very-high copy number nature. “This characteristic is associated with the presence of a single-point mutation in the plasmids’ origins of replication and further derepresses plasmid replication, leading to up to a five-fold increase in plasmid titers,” he explains.
Leaky promoters that lead to pre-mature protein expression during the growth phase can also be detrimental to upstream plasmid success, Varadi comments. He adds that some large pDNA losses can also occur during alkaline lysis. In fact, a well-defined alkaline lysis step is far more important in reducing plasmid loss than avoiding cyclic performance during downstream chromatography operations, he contends.“The lysis step has several critical parameters that need to be well balanced, like buffer volumes, buffer contact time time, and applied shear forces during mixing,” Varadi explains.
Plasmid DNA fermentation and cell lysis are complex processes that can be influenced by many different process parameters. Both plasmid titer and quality, including supercoiled plasmid content, can be directly affected, according to Santos. “Proper control of plasmid replication repression and induction is key to reach high volumetric and specific yields. In the fermentation process, this is achieved through utilization of appropriate culture media, robust control of the bacteria growth rate, appropriate temperature set points, and the availability of oxygen,” she says. For cell lysis, Santos stresses that it is crucial to recover as much material from the E. coli biomass as possible without degrading the product. “A fine and well-balanced alkaline lysis approach is the preferred unit operation to meet this objective,” she observes.
There are several steps that can be taken to boost efficiency and productivity of pDNA fermentation and lysis processes. During cell-line development, reducing cell stress in general improves the quality and quantity of pDNA produced, according to Varadi. During fermentation, reduction of metabolic burden and the use of well-designed fed-batch profiles is highly beneficial, as lowering metabolic burdens leads to higher product yields.
Closer real-time monitoring of critical parameters during batches, such as online cell density measurements, and comparison of multiple production runs both play an important role in improving plasmid fermentation processes, according to Shankar. “Implementation of process analytical technology tools for various critical process (off-gassing) parameters in combination with at-line analysis of product parameters (plasmid content and homogeneity) helps to unravel hidden impacts of process variables,” he says.
One thing Varadi would like to see is the introduction by device suppliers of more scale-down (e.g., lab-scale) versions of their equipment. “Performing process development in an optimally scaled-down process makes the upstream process more reliable and robust when scaling up,” he contends.
The successful development of platform manufacturing processes for recombinant proteins and monoclonal antibodies has created the desire to establish similar platform solutions for other biologic materials, including pDNA.
“The aim is to find one process that would fit all, rather than optimizing each process for different plasmids. This would offer cost and timeline advantages, ultimately accelerating the delivery of treatments to patients,” Craig remarks. “Operating a robust plasmid production platform that facilitates the transition of programs from a pre-IND [investigational new drug application] stage towards the clinic and to market can lead to the delivery of the required product quantities with the expected purity profiles, thereby greatly accelerating development timelines,” he adds.
That is not so easily done, however. “Development of platform processes are to a certain extent possible but there cannot be a magic bullet for every type of plasmid,” states Schmeer. “Individual plasmid size and sequence can have a huge impact on upstream process efficiency, which is often not fully appreciated,” he adds.
Output variability from platform processes, Santos agrees, is mostly associated with the specificities of the plasmid sequences themselves. “Large constructs tend to be lower yielding, and plasmids carrying repeated sequences such as ITRs and polyA tails have been documented to be more prone to deletion and recombination events,” she notes.
The solution, according to Bowen, is to develop a “flexible” platform by investigating a variety of plasmid types (size, complexity, etc.) and E. coli strains during process development to determine their impact on plasmid yield and quality. This will allow for flexibility when manufacturing subsequent plasmids.
Adding optimization flexibility to a robust platform process facilitates the delivery of plasmid quantities, including meeting quality specifications for challenging plasmids, Craig agrees. He also points out that timelines can be further accelerated by using off-the-shelf plasmids. “Having a well-characterized set of plasmids in advance, such as pHelper and pREPCAP plasmids, will save valuable time on subsequent applications, particularly when used in combination with a platform process,” he states.
For Varadi, developing a platform process for upstream plasmid manufacturing is not only possible, but mandatory. “A well-designed platform embraces many plasmid types and enlarges the spectrum of potential customers while reducing manufacturing costs,” he comments. One key to success, Varadi adds, is size optimization of plasmids by reducing “junk” DNA stretches within the backbone and the gene-of-interest cassette. He also notes that a combination of host-cell and plasmid-backbone mutations (already characterized in literature) is beneficial for copy-number increases that should be evaluated for industrial purposes.
There is no doubt that the demand for plasmid DNA will continue to increase in the coming years. All therapy types that require pDNA as a raw material are expanding massively and require an industry in the background producing them, Varadi contends.
“Over the next few years, high-capacity manufacturing will not be the only prerequisite for pDNA manufacturers; they will also need to have the ability to produce a multitude of different constructs in smaller quantities,” Varadi observes. “High-capacity manufacturing will be necessary to meet demand, but that cannot be achieved only through upstream process optimization. Wide implementation of single-use technology and simplified scalability solutions as a link from non-GMP process development laboratories to GMP suites will be necessary,” he insists.
Scale and quality will indeed be the two key aspects that must be addressed for future pDNA manufacturing, according to Shankar. He agrees that “GMP production of plasmids through the adoption of single-use technologies for upstream processing will turn the tide of manufacturing for novel advanced therapy medicinal products.”
Cynthia A. Challener, PhD, is contributing editor to BioPharm International.
Vol. 36, No. 3
Pages: 10–13, 36
When referring to this article, please cite it as Challener, C.A. Achieving High Yields in Upstream Processing of Plasmids. BioPharm International 2023 36 (3).