In recent times, the need for small amounts of plasmid DNA that is clinical grade has increased. To meet this demand we developed and tested an adapted Good Manufacturing Practices (GMP) manufacturing method that is able to make small batches (1-4 milligrams) of the mRNA plasmid.
The new method doesn’t require any sophisticated equipment for production. It makes use of only production materials that are disposable. That makes it easy to use and reduces the need for line clearance. We have been able to successfully use this method to make tiny batches of distinct plasmids.
The plasmids produced that are both created within the Electroporation buffer, get combined and diluted in small, single-use portions.
Quality control has confirmed the efficacy of the method. A stability analysis also proved that the final product can be used for a minimum of two years.
The final formulation for patients will then be utilized in the phase I/II clinical trial. In this step, the retina cells of patients suffering from Age-Related Macular Degeneration, are transfected.
The method of GMP plasmid production described could be used in a general way for other plasmid constructs as well as final formulations.
Gene therapy as well as (stem) cells have emerged as appealing treatment options for many different illnesses. In addition to the widely used virus vectors, non-viral delivery techniques using plasmid DNA are particularly intriguing.
They tend to be cheaper and quicker to create. In clinical applications, Good Manufacturing Practice (GMP) quality DNA plasmids are required. We’ve previously presented methods to make large-scale (>100 milligrams) batches of plasmid DNA that was used for DNA vaccination purpose at the institute (Quaak and co. (2008); Samuels et al. 2017).
In recent years, the need for small amounts of plasmid DNA for Phase II and III trials has grown. In addition, clinical studies of phase I/II small aliquots of plasmid could also be beneficial for animal experiments. There are also pilot studies where only a small amount of DNA is required.
In this regard, an extremely small-scale manufacturing procedure was designed. This production process is a scaled-down variant of a previously reported GMP production method (Quaak and co. 2008) and can give small amounts (1-4 milligrams) of DNA plasmids.
This production technique offers two advantages in comparison to the larger-scale process. One of which is that it doesn’t require any specific equipment for production like chromatography or bioreactor.
The entire material is disposable, eliminating any cleaning testing, which will save more time and money. Utilizing a small-scale as well as disposable Tangential Flow (TFF) process to focus plasmids and exchange solvents there are no organic solvents needed.
The small-scale plasmid DNA production method was subsequently used for the production of two plasmids, pFAR4-CMV-SB100x-SV40 and pFAR4-ITRs-CMV-PEDF-BGH.
They are miniplasmids devoid of an antibiotic resistance gene. A mix of the two plasmids is currently being developed for the ex-vivo electroporation of retina cells. It is done as part of the course of a clinical trial. Two plasmids (pFAR4-PEDF and pFAR4 SB100x) are created independently in two bulk plasmids.
They’re blended in a 1:16 ratio. Then aliquoted into 25 ml vials in glass as a final, single-use patient-specific formulation in aseptic conditions. From the final formulation, two clinical batches and a pilot batch were created.
It proved the reliability and stability of the manufactured product. The pharmaceutical product (investigational medicine) will be utilized in the phase I study of patients suffering from macular degeneration.
2. Materials and methods
2.1. General
The plasmid DNA was created and prepared under aseptic conditions in two cleanrooms of grade B (Interflow, Wieringerwerf, The Netherlands). One cleanroom is designed for the handling of bacteria (upstream processing) and the other one is used for the purification of plasmids and formulating the products (downstream treatment).
Both cleanrooms are equipped with a Grade biosafety cabinet, in which every critical procedure was carried out. Both cleanrooms are subjected to surveillance programs for viable and non-viable particles at both operating and resting states.
The entire process was performed in accordance with Good Manufacturing Practice (GMP) guidelines (EudraLex (2019)). In the entire manufacturing and formulation procedure, only disposable items were utilized.
Pharmaceutical excipients, buffers, and the primary packaging ingredients used in the manufacturing of plasmids were European Pharmacopeia (Ph.Eur.) grade (if feasible) and were accompanied by a Certificate of Analysis (CoA) from the vendor. All excipients and materials were certified on the basis of internal quality control as well as the vendor’s accreditation.
All buffers for bacterial elimination as well as pre-clarification and purification were made in the production facility within the pharmacy department of the Antoni van Leeuwenhoek-The Netherlands Cancer Institute (AvL-NKI).
The Electroporation Buffer that was used for the final formulation was provided by 3 P Biopharmaceuticals (Navara, Spain) and was created in the FP7 EU TargetAMD consortium.
This iso-osmotic buffer was developed for electroporation in vivo of retina cells and was evaluated for sterility as well as endotoxins. Other buffer compositions were provided by Qiagen and included in Table 1.
Gene therapy as well as (stem) treatment with cells has been attractive treatments for many ailments over the past few years (Ginn and colleagues. (2013) Gorabi et al. (2018); Melero et al. 2014).
In addition to the widely used viral vectors, non-viral delivery techniques using plasmid DNA are particularly intriguing, as they tend to be less expensive and quicker to create. For use in clinical settings, Good Manufacturing Practice (GMP) quality plasmid DNA is needed.
We’ve previously presented methods to make large-scale (>100 milligrams) batches of plasmid DNA which we used for DNA vaccine purposes at the institute (Quaak and co. (2008); Samuels et al. 2017).
In recent years, the need for small amounts of plasmid DNA to conduct phase II and III trials has grown. In the clinical phase, I/II small aliquots of plasmid are also useful for animal studies and pilot studies, where just a few ml are needed.
Thus, an extremely small-scale manufacturing method was devised. This production process is a smaller version of a previously reported GMP production method (Quaak and co. (2008)) and can give small amounts (1-4 milligrams) of DNA plasmids. This method of production offers two advantages in comparison to the original method which was on a larger scale.
It doesn’t require any specific equipment for production, for example, the bioreactor or chromatography equipment 2) the materials are all disposable and do not require any cleaning testing, which will save extra time and money.
With the use of a small-scale as well as disposable Tangential Flow (TFF) technique to focus plasmid and exchange solvent there are no organic solvents needed.
The small-scale plasmid DNA production method was subsequently used for the production of two plasmids, pFAR4-CMV-SB100x-SV40 and pFAR4-ITRs-CMV-PEDF-BGH, which are miniplasmids devoid of an antibiotic resistance gene. A mix of the two plasmids is currently being developed for the electroporation in vivo of retina cells as part of the course of a clinical trial. Both plasmids (pFAR4-PEDF and pFAR4 SB100x) are made separately in two separate bulk plasmids.
They are blended in a 1:16 ratio and then aliquoted into 25 ml vials in glass for a single-use patient-specific formulation with aseptic treatment. The final formulation was then created. An experimental and clinical batch was created that confirmed reliability and stability of the manufactured product. The pharmaceutical product (investigational medicine) is expected to be used in a Phase I study of patients suffering from macular degeneration.
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