Quality by Design for recombinant Adeno-Associated Viruses
Introduction
Recombinant Adeno-Associated Viruses (rAAVs) are genetically modified viruses (Adeno-Associated Viruses) that act as vectors to deliver a therapeutic gene to a target cell. They are Advanced Therapy Medicinal Products (ATMPs) as defined by the Regulation 1394/2007 and Part IV of Annex I to Directive 2001/83/EC. The gene is incorporated into the target cell’s nucleus and remains there as stable episomes that do not integrate into the host cell’s deoxyribonucleic acid (DNA); the fact that no integration occurs means that compared to other gene therapy medicinal products (GTMPs) the risk of insertion into proto-oncogenes by rAAVs is avoided; this combined with the low pathogenicity of the wild type AAVs, makes rAAVs the desired choice for developing GTMPs and they are one of the most widely applied vectors for gene therapy and vaccines.
After the rAAV infects the host cells, the transgene is constitutively expressed; the extent of expression is dependent upon the cellular environment and the design of the transgene. Genetic expression produces the protein(s) of interest that are required for the therapeutic effect. In any particular disease / condition (for example if a patient has a genetic mutation such that they are unable to correctly produce the protein (e.g. point mutation(s)), the patient is incapable of producing the relevant protein at all, or enough of it), the gene therapy approach of rAAVs can provide the necessary gene(s) that encodes for the protein(s) in question and produces the therapeutic benefit.
Quality by design
Quality is defined as the suitability of a medicinal product to perform its intended function. The pharmaceutical form and the formulation of the final medicinal product are derived from the Quality Target Product Profile (QTPP). The QTPP is derived from the Target Product Profile (TPP); the TPP includes information on, for example, the clinical indication and the patient population.
The QTPP is a prospective summary of the desired specification to ensure the medicinal product’s quality, safety and efficacy. Product development studies includes vector design, codon optimised gene insert, excipient compatibility, prior knowledge and experience with other rAAVs, literature and published articles on other similar products, and stability studies as appropriate. Once the formulation and dosage form has been decided upon, the next stage is to determine a manufacturing process that can consistently produce the medicinal product with the desired functionalities; i.e., the desired quality of the medicinal product to be used in non-clinical and clinical studies. The data obtained from these studies can be used to apply for a Marketing Authorisation (MA).
Quality by design (QbD) is the process of using a risk based approach, (including theoretical and scientific thought based rationale, experimental assessments: design of experiment and accumulation / research for knowledge around the medicinal product or similar medicinal product/modalities), to design a manufacturing process that can consistently produce the rAAV medicinal product of a desired quality. QbD means that the quality of the final rAAV drug product is built into the manufacturing process instead of confirmation through testing.
The ICH guideline (ICH Q8 Pharmaceutical development (R2)) provides the information and regulatory expectations for QbD and this should be referred to for more information.
Manufacturing development of rAAVs
Manufacturing processes documented and published in my research for rAAVs seem to be based on an upstream process (USP) and a downstream process (DSP).
The USP involves the manufacture of a bulk containing the rAAV in a ‘broth’ of other materials; the rAAVs are harvested from the bulk solution for purification. The DSP consists of the purification and concentration of the rAAV and a platform approach to DSP is usually applied.
A platform is where information on the technology of the manufacturing process of another similar product can be leveraged to the proposed product. For DSP there appears to be a standardised approach used in industry with regards to the purification techniques and steps.
For rAAVs, the USPs that I have located are detailed in the table below. The methods described are triple transfection methods using plasmids and transfecting agents and infection methods using either Baculovirus or Adenovirus in host cell lines. These techniques produces a bulk harvest. Important factors are the quality and purity of the host cell line; for rAAV drug products for human use, cells are expanded from qualified cell banks since the quality of host cell line can impact on the quality of the final rAAV drug product.
The bulk harvest containing the viral particles is usually treated with endonucleases (either at the point of harvest or afterwards) to break down any host cell nucleic acids or unused nucleic acid which may present as impurities in the final drug product. This bulk harvest is then purified in the DSP.
I have been able to find another USP processes, this involved establishing rAAV packaging cell lines with the production of rAAV vectors using recombinant Herpes Simplex Virus (HSV); however, so far I haven’t got around to determining how well established this manufacturing procedure is in industry; HSP has low pathogenicity so this is a viable alternative...maybe that is something for me to investigate.
In relation to QbD; the aim is to optimise a manufacturing process to consistently deliver a defined quality medicinal product and therefore it is important to identify specific manufacturing operational steps, actions or materials that could impact on the quality of the rAAV drug product. A risk assessment of each step (cell expansion/cell lysing/purification/other manipulation techniques), should be performed to determine the impact on quality and ultimately the critical process parameters (CPPs).
Before any of this can happen though, the quality attributes to measure have to be identified. Proposed quality attributes would be ‘preliminary’ at first and the criticality of each attribute is determined by applying risk assessment techniques to the preliminary attributes. This involves determining the risk associated with changes in a particular attribute, the likelihood of this risk/hazard occurring and the severity of the risk or hazard in humans. The other thing that needs to be taken into consideration when making these assessments is the level of uncertainty associated with each risk. With rAAVs there could be limited information compared to conventional medicines; trending data is not necessarily as comprehensive compared to traditional conventional medicines. The application of scientific thought based rationale or theoretical approaches, such as using wild types approaches to extrapolating to the concerned product, or using literature sources and available prior knowledge is a good starting point. Next stage is risk scoring, then risk ranking to give the preliminary CQAs.
The rAAV functional titre, product-related and process related impurities, and adventitious agents are attributes that can impact on the quality, safety and efficacy of the product; these attributes should be explored through the risk assessment described above to determine their criticality and to decide on the required control methods and testing strategy.
Product related impurities consist of:
Noninfectious AAV
Deamidated AAV
Aggregated AAV
Empty capsids
Encapsidated host cell DNA
Encapsidated helper DNA
Replication competent rcAAV
Process related impurities include:
Residual host cell DNA/RNA
Residual host cell protein
Residual plasmid DNA
Residual helper viruses
Residual animal-derived cell culture medium components
Detachment enzymes
Detergents/Leachables/Nuclease
The CPPs are established by assessing the impact of varying the process parameters on each of the CQAs. Consideration of the quality of the raw / starting materials is a pre-requisite; control measures should be in place to mitigate negative impact on poor quality raw / starting materials on the CQAs of the drug product.
It is important to note that there are ‘obligatory CQAs’ these should always be assessed using standard techniques for GTMPs; these include the adventitious agents, but also genetic identity, genetic stability, potency, purity and finished product stability.
Needless to say, for rAAVs used in clinical trials and for marketed medicinal products Good Manufacturing Practice (GMP) is a requirement. Materials for non-clinical studies, are not required to be manufactured to GMP, however, the manufacturing process should be well defined and comparable to that proposed for the clinical trials.
In regard to adventitious agents, a revised GMP on the ‘Manufacture of Sterile Medicinal Products’ is coming into force on 25th August 2023; the design space for rAAVs needs to include a Contamination Control Strategy (CCS); rAAVs drug products manufactured in aseptic, sterile environments will be inspected against this new GMP, so that is something to bear in mind, since the design of manufacturing facilities will need to be optimised using a QbD approach.
Once all of the above has been determined, then the design space that includes the Normal Operating Ranges (NOR) can be established; the establishment of the design space enables a better regulatory approach and health authority flexibility, with less requirement for post approvals changes in marketed product.
The design space has to be validated; this means that excursions outside of the NOR do not require further regulatory approval since design space has already been established and confirms the quality. This doesn’t obviate the manufacturer from conducting a full investigation to determine the reason(s) for the excursion and to monitor future excursions. However, for the urgent need of Advanced Therapy Medicinal Products (ATMP) this approach enables the despatch of potentially life saving medicines to patients who need them.
I noted from a seminar that I attended that regulators (specifically the FDA) for ATMPs are requesting a validated design space as part of the application for a licence; they require the applicant to take into consideration the total patient population for the disease. ATMPs are challenging with regards to scaling up and scaling out, hence the request. These considerations do have to be balanced out against the limited timelines for manufacturing development, particularly those taking accelerated routes to market. Manufacturing development using the concepts and processes of QbD is an optimal risk based approach to ensure consistent quality products are available to patients who urgently need them.