The demand for DNA vaccines for gene therapy, vaccination and for the treatment of diseases such as cancer, malaria, swine flu, HIV, melanoma, etc. is on the increase (Prather et al., 2003; Williams et al., 2009). This is because DNA vaccines triggers cellular and humoral immune responses, safe and stable (Prather et al., 2003). Therefore, there is need to develop manufacturing facility for DNA vaccines production to meet the rising demand. However, the design, operations and layout of the manufacturing facility must conform and comply to standards, specifications and guidelines stipulated by regulatory authorities such as the U.S. Food and Drug Administration (FDA), Medicines and Healthcare products Regulatory Agency (MHRA), European Medicines Agency (EMA), World Health Organisation (WHO) and the regulation of the country in which the facility is to be constructed. In addition to meeting this regulations and guidelines the DNA vaccines production process, design and premises of its manufacture must conform to good design practices (GDP) and current good manufacturing practices (cGMP) (Shamlou, 2003; Przybylowski et al., 2007).
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The commercial scale production of DNA vaccines is justified by economics/cost, health, safety and environment, compliance to legal standards and production under Good Manufacturing Practices (GMP) (Shamlou, 2003). This is to ensure that manufacturing processes are controlled and performed according to design specifications and operational procedures in order to ensure that quality is built into the product (DNA vaccines) to assure safety, efficacy, purity and identity consistently (Przybylowski et al., 2007). In addition, GMP requirements are open ended, however the International Society of Pharmaceutical Engineers (ISPE) has enumerated the principal steps to current GMP which include operational procedures, qualification and validation of process performance, design, quality control testing, adequate process control, sterilization in place (SIP), cleaning in place (CIP), layout design, quality management, documentation and audit of facility as necessary to ensuring specification and maintenance of product identity and compliance to regulations (WHO, FDA, MHRA, etc.) and current good manufacturing practices (cGMP) (Day, 2004).
The issue of location for the manufacturing facility is crucial to its profitability as it is influenced by raw material supply, transportation, utilities, environmental impact, waste disposal, local community considerations, personnel, climate, plant size and availability of land (Sinnott, 2005). Moreover, before the design and installation of a new facility for pharmaceutical and biopharmaceutical product manufacture, an environmental impact assessment (EIA) is perform and approved (Davda, 2004). Hitherto, the design of any manufacturing facility must integrate the design of a treatment process and safe disposal of the waste generated to specified legal standards by regulatory authorities and eliminate/minimise harm to health and safety of personnel, environment and product contamination. The manufacturing facility layout must be designed to aid good raw material flow, waste flow and personnel flow around the factory to reduce risk, cross contamination and ensure that production activities and factory operations are performed smoothly and follow a defined procedure. The pharmaceutical manufacturing process must be conducted in clean environment and clean room in which the temperature, pressure and humidity are controlled to specified conditions by regulation (U.S. FDA, WHO, ISO, MHRA, etc). All these are the component of current Good Manufacturing Practices (cGMP) to build quality assurance, consistency and safety of therapeutic product (DNA vaccines) to human life (Signore and Terry, 2008). The entire operations and activity should be performed by trained and competent personnel and quality management for a satisfactory quality assurance (QA/QC).
Aims and objectives
- The defined goal of this project is to develop a detailed design and layout of a manufacturing facility for the production of DNA vaccines for commercial scale, applying current Good Manufacturing Practices (cGMP) and in compliance to regulatory guideline (FDA, FDA, MHRA, WHO, etc.).
- Provide detail methods for qualification and validation of the designed process, performance, quality control and enumerate the personnel/staff involved in the project.
- Estimate the timescale of the project.
DNA vaccines production mainly starts on a bench scale through pilot scale to large scale production (Ferreira et al., 2000; Bequette et al., 2004). The design of a large scale facility for the manufacturing of DNA vaccines involves the selection of suitable plasmid DNA constructs/vectors (ColE1-type vectors, pUC vectors, pBR322 plasmid vector, etc.) that will replicate at high copy numbers, the production microorganism cell bank (Escherichia Coli), subsequently followed by fermentation process in the bioreactor under optimum conditions and control media (temperature, pH, pressure, etc.) to maximise cell growth, cell lysis to break the cells to release the DNA, isolation by precipitation of genomic DNA, cell debris, proteins and RNA, purification by anion exchange chromatographic technique because DNA is negatively charged, formulation and blending, sterile filling, packaging and storage in the fridge (Ferreira et al., 2000; Prather et al., 2003; Przybylowski et al., 2007).
The process stages:
- Raw material preparation
- Media preparation
- Working cell bank
- Bioreactor for cell growth under controlled temperature, pH and pressure
- Buffer capacity help to stabilize pH of the media
- Cell lysis
- Clarification and concentration
- Primary purification by ion exchange chromatography
- Secondary purification by size exclusion chromatography
- Formulation and blending (conversion of bulk product into usable form)
- Sterile filling and packaging.
Process flow sheet
Always on Time
Marked to Standard
The process flow sheet shows the interconnection of the various unit operations and the process stages involved from the raw materials to the DNA vaccines. Each pieces of equipment in the process flow sheet should be designed to conform and comply with standard and code of practice of either International Organisation for Standardization (ISO), British Standard Institution (BSI), American Petroleum Institute (API), American Society for Testing Materials (ASTM), American National Standard Institution (ANSI), etc. to ensure safety, selection of suitable material of construction, and also equipment manufacturers work to produce facilities according to standardized design and size (Sinnott, 2005).
The various unit operations shown on the process flow sheet in Fig.1.2 above and the ancillary buildings required to support the manufacturing facility for DNA vaccine production are laid out to give an economical flow of raw materials to final product storage, flow of personnel and waste around the production site to conform to Good Manufacturing Practice (GMP), reduce risk and product contamination (Sinnott, 2005; Signore and Terry, 2008). Clean rooms, waste treatment area, hazardous process and raw materials must be isolated and arranged for safety and cross contamination of product. The ancillary buildings and support services required for the manufacturing facility include:
- Storages for raw materials and DNA vaccines.
- Laboratories for quality control.
- Maintenance workshops and warehouse.
- Utilities: steam, compressed air, power generation, refrigeration, water (WFI), CO2, N2 etc.
- Cleaning in place (CIP) and Sterilisation in place (SIP).
- Effluent treatment and disposal plant.
- Process control room
- Administrative offices
- Fire stations and other emergency services
Amenities required include:
- Roads and car parks.
- First aid centre.
- Rest room, changing room, training room and visitors centre.
Plant layout for production area
The detailed design and layout of the DNA vaccines production rooms and equipment is designed to minimise risk, reduce cross contamination, permit effective cleaning and sterilisation of external and internal surfaces of process equipment by the use of clean in place (CIP) and sterilisation in place (SIP), enhance maintenance and control of clean rooms temperature, pressure and humidity under standard operating procedures (SOPs) (Przybylowski et al., 2007). The facility layout also considered that the design of the production rooms, equipment and the flow of materials and personnel are known to have key impact on manufacturing cost, operational procedures and productivity (Drira et al., 2007). The DNA vaccines manufacturing facility layout design is shown in FIG. The efficiency and safety of the production environment and manufacturing process are dependent on the layout of the facility (Jacobson et al., 2002).
The raw materials, reagents and utilities required for the DNA vaccines manufacturing facility are: plasmid DNA vectors, nutrients, glucose, water for injection (WFI), sterile air, salt, buffer capacity (to stabilise pH of fermentation), liquid nitrogen N2, and antibiotic, alkaline and working cell banks. These are placed in the quarantine storage room and undergo quality control testing to ensure that specification are met before used for DNA vaccines production for quality assurance (QA). The flow of materials from the raw materials to the final product (DNA vaccines) is shown in FIG. above and the final DNA vaccines products are stored in a sterile room in a freezer at -20oC (Przybylowski et al., 2007).
The compliance to current Good Manufacturing Practices (cGMP) and regulatory guideline depends on people. It is essential when developing new facility to integrate all relevant personnel from production, logistics, quality control and engineering in the inception phase of the design. Therefore, for a satisfactory quality assurance of the DNA vaccines production and facility design layout, the following trained professionals and qualified personnel are employed: chemical engineers, microbiologist, pharmacist, civil engineers, project manager, mechanical engineers, electrical engineers, architect, etc. to carry out defined tasks and responsibilities.
Qualification and validation
The qualification and validation of pharmaceutical manufacturing facilities at regular intervals is an integral part of good manufacturing practices (GMP). This is a documentary evidence that assures that the DNA vaccines production facility is performing satisfactorily and consistently to specification for the intend purpose (Day, 2004). Qualification and validation is required for the DNA facility are: design qualification (DQ), installation qualification (IQ), operational qualification (OQ) and performance qualification (PQ) (Day, 2004; Chaloner-larsson et al., 1997).
Design qualification (DQ)
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Design qualification is carried on the following production pieces of equipment of the manufacturing facility such as bioreactor, centrifuge, ion exchange chromatography, size exclusion chromatography, microfiltration system, ultra-filtration system and lyophilizer, for verification and documentation as a prove to show that equipment conforms to regulatory standards such as ISO 9000, BSI, etc.
Quality control of the DNA vaccines is one of the key component of current good manufacturing practices (cGMP) and regulatory guideline of U.S. FDA, WHO, MHRA, ISO 9000 etc. It involves testing procedures employed to ensure that DNA vaccines product and raw materials used for its production meet the specification, quality and standard. The testing assays required in quality control room are:
- High performance liquid chromatography (HPLC) - to determine the percentage of RNA, supercoiled and nicked.
- pH meter - test for residual buffer salts and alkaline.
- Agarose gel electrophoresis (AGE) - test for plasmid DNA vaccine purity, determine RNA and genomic DNA presence in the product.
- Gas chromatography - test for the presence of ethanol, determine plasmid size
- Flame ionization detector (FID) - test for the presence of isopropanol in the product.
- Transfection/Immunofluorescent staining - test for potency of plasmid DNA vaccines.
- Kinetic chromogenic limulus amoebacyte lysate (LAL) - test to quantify the presence of endotoxin in the product
- Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) - test for the quantity of proteins in the product (DNA vaccines).
- GeneQuant spectrophotometer - test to quantify the purity of the DNA vaccines product.