How to do quality control in today's rapid development of gene therapy products?

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Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities, thereby achieving the purpose of treatment, that is, inserting exogenous genes into appropriate recipients of patients through gene transfer technology In cells, products that express foreign genes can treat certain diseases

Broadly speaking, gene therapy can also include measures and new technologies to treat certain diseases at the nucleic acid level, such as RNA interference technology

In May 2019, the U.

Gene therapy product approach

Gene therapy products are being studied for the treatment of diseases including cancer, genetic diseases and infectious diseases

There are two ways of gene therapy in vivo and in vitro.

Plasmid DNA: Circular DNA molecules can carry therapeutic genes into human cells through genetic engineering

Viral vectors: Viruses have the natural ability to deliver genetic material to cells, so some gene therapy products are derived from viruses

Bacterial vectors: Bacteria can be modified to prevent them from causing infectious diseases, and then used as carriers (vectors) to carry therapeutic genes into human tissues

Human gene editing technology: The purpose of gene editing is to destroy harmful genes or repair mutant genes

Patient-derived cell gene therapy products: cells are removed from the patient, genetically modified (usually using viral vectors), and then returned to the patient

Figure 1 Gene therapy product approach

Quality research

With the deepening of product knowledge and the development of testing technology, product quality research should continue to be supplemented and improved throughout the entire life cycle

The research of quality characteristics should select representative process batches and samples of appropriate production stages as the research objects.

Quality research content should cover all characteristics that may be related to product safety and effectiveness, generally including structure, identification, general physical and chemical properties, purity, biological activity, gene transduction efficiency, impurities, genotype, phenotype, etc.

▲Structure and characteristic analysis

The study of structure includes primary structure and high-level structure

▲Biological activity

Biological activity research and methodological establishment should be developed based on product indications, route of administration and mechanism of action, and as far as possible the establishment of body, internal and external analysis methods that are the same or similar to the mechanism of action for activity research and quality control

▲Analysis of purity, impurities and contaminants

Impurities mainly include product-related impurities and process-related impurities

Product-related impurities include all non-target or non-functional production products
.
Viral vectors generally should analyze the residual levels of impurities in the product such as empty shell viruses, mispackaged viruses, hybrid viruses, inactive virus particles, and virus aggregates in the product, and evaluate their safety; nucleic acid vectors are generally The residual levels of error sequences, incomplete sequences, degraded fragments, differential structures, incorrect modifications, or misassembled components of complex delivery systems should be analyzed; microbial vectors generally deal with the monoclonality of strains, the loss rate of plasmids or modified genes, etc.
Perform verification
.

▲Content

Gene therapy products establish physical quantity and the number of detected biological indicators, such as: the number of particles, the infectious titer or number of infectious particles, genomic DNA / RNA or plasmid DNA concentration, the number of bacteria and the like
.
Viral vectors should control the ratio of the total number of particles to the infectious titer or the number of infectious particles.
For viral vector preparations, it is recommended that the physical titer be used to calculate the clinical measurement
.
The viable microbial carrier should control the rate of viability and the ratio of viable bacteria to dead bacteria
.
As far as possible, the analysis should use standard or reference substances to calibrate the assay results
.

▲Analysis of other characteristics

Usually specific physical and chemical analysis, such as appearance, clarity, visible foreign matter, insoluble particles, pH and osmotic pressure
.
In addition, the replication capacity of the viral vector, the insertion site, and the transduction rate of the plasmid vector may also be required
.

QC

The formulation of quality standards is for the purpose of controlling the quality of the final product and batch-to-batch consistency.
Specifically, quality standards should be formulated for samples at different stages according to process and control requirements, which generally include quality standards for stock solutions, semi-finished products (if any) and preparations
.
The determination of the quality standard should be based on quality research, and the specific content of the quality standard should be determined according to the correlation between product quality attributes and safety and effectiveness, which generally include inspection items, analysis methods and acceptance standards
.

Standard limits should generally be set based on the setting of target product quality, statistics of representative process batch analysis data, stability research results, and human or animal safety research data
.

The quality standard of the stock solution generally includes appearance, identification, physical and chemical properties, purity, content, activity, exogenous factors, endotoxin, impurities (product-related and process-related impurities), and other test items prescribed by the Pharmacopoeia
.

For non-replicating or conditionally replicating virus vectors, replicable viruses should be detected and controlled
.
In addition to the above-mentioned original solution testing items, the preparation quality standard should also pay attention to other quality attributes affected by the preparation prescription, preparation production process and packaging container, and generally include (but not limited to): transduction efficiency, infectious activity, preparation appearance, and filling volume , Extractable volume, water residue, physical and chemical properties of the preparation (such as pH, refractive index, osmotic pressure, insoluble particles, visible foreign matter, etc.
), content of key excipients (aluminum, compound excipients, etc.
), preparation process impurities, replicable viruses ( If appropriate), and the preparation inspection items required by the Pharmacopoeia
.

If the preparation uses a special container or a combination device of medicine and equipment, a specific release test needs to be added according to the function of the device
.
For some test items that are not included in the quality standards, the rationality should be explained and the basis for verification should be provided
.

Stability study

The stability study of gene therapy products can be carried out with reference to the "Technical Guidelines for Stability Research of Biological Products (Trial)" and the general principles and related requirements of ICH Q5C.
At the same time, the research plan should be rationally designed according to the characteristics of the product and the status of clinical use
.

Research projects generally include long-term stability, accelerated stability, influencing factors research, transportation stability, use stability, etc.
The research conditions should be specifically determined according to the specific storage, transportation and use conditions, and the significance of the research results under the corresponding research conditions
.
The research should select representative process samples, packed in representative packaging containers or using equipment
.

Stability research and monitoring projects should be comprehensive, especially testing projects that have important indications for product safety, effectiveness, and stability
.
The study period should cover the actual storage or use period
.

Container and closure system

Containers and closed systems generally include packaging containers for raw liquids, semi-finished products (if any), preparations, storage containers for raw materials and intermediate products during the production process, and production pipelines that directly contact the product during the production process, such as biological reaction bags and disposable pipelines.
and so on
.

In order to avoid unintended effects of storage containers or closed systems on the quality of products, compatibility studies, airtightness studies, and safety assessments should be conducted on the containers and closed systems, and airtight storage containers under specific conditions should also be applicable under corresponding conditions research, such as frozen adaptability
.

For secondary packaging materials with special functions, the secondary packaging materials should be included in the research of containers and closed systems, and their corresponding functions should be studied and verified
.
If special drug delivery devices are involved, such as electroporation devices, nasal spray devices, needle-free syringes, etc.
, relevant research materials or other applicable supporting materials must be submitted
.

References:

[1] Technical guidelines for pharmaceutical research and evaluation of gene therapy products (draft for comments)

[2] Overview of China's gene therapy industry in 2021