June 24, 2022

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IND/NDA pharmacy evaluation for gene therapy products application

IND/NDA pharmacy evaluation for gene therapy products application


IND/NDA pharmacy evaluation for gene therapy products application.  Compared with other viral vectors, recombinant adeno-associated virus (rAAV) vectors have the advantages of strong infection ability, sustainable expression of target genes, non-pathogenicity and non-genomic integration, etc., and has become the main viral vector for gene therapy in vivo.

This article summarizes the latest research progress of rAAV gene therapy products, and discusses the key points of pharmaceutical evaluation of such products from the aspects of raw materials, production technology and quality control, in order to promote the clinical transformation and application of such products. At the same time, the pharmacy evaluation consideration points and common problems of 3 foreign marketed products are discussed.


The development status of rAAV gene therapy industry

According to incomplete statistics, at least 238 rAAV gene therapy products are currently undergoing clinical trials, and 3 rAAV gene therapy products have been registered in Europe and the United States (Table 1).

Table 1 Clinical application of rAAV gene therapy products
IND/NDA pharmacy evaluation for gene therapy products application



1. Research content and evaluation of raw materials

1.1. Virus molecular design

The vector design of rAAV should be based on clinical effectiveness and safety, generally targeting specific tissues or cells, deleting genes related to virulence, pathogenicity or replication ability to ensure the safety of the product. At the same time, the design should consider the size of the vector genome, packaging efficiency and expression efficiency, and minimize the homology with related viruses in the body to avoid the formation of replicating viruses. The risk of introducing antibiotic resistance genes should be considered when constructing virus plasmids. Generally, ampicillin resistance genes should not be used.

1.2, production cell matrix

At present, the cell matrix used in the production of rAAV in the industry includes mammalian cells (HEK293, BHK), human tumor cell lines (HeLa, A549), and insect cell lines (SF9).

Packaging cells should be fully identified and a bank should be established. Identification items generally include identification, purity, genotype/phenotype, tumorigenicity/tumorigenicity, genetic stability, and introduced sequence. In addition to conventional sterile, fungi and mycoplasma, special attention should be paid to species-specific viruses in the cell matrix. For example: HEK293 cells should be tested for CMV, HIV-1/2, HILV-1/2, HH-V6/8, JV virus, BK virus, EBV, parvovirus B19, HBV, HPV and HCV and other viruses; SF9 cells should be tested for spirogen Insect viruses such as human body, rhabdovirus.

When using human tumor cell lines as packaging cells, the risk of tumorigenicity and tumorigenicity, as well as the infection of tumorigenic viruses, etc., should also be fully assessed. For example, HeLa cells are tumor cells derived from human cervical cancer tissues, which are known to contain 50 copies of HPV-18 genome and virus E6/7 gene sequence. Therefore, it is recommended to check the transfer gene residues in the quality control project.



2. Research and evaluation on production process

2.1. Upstream process of AAV products

2.1.1. Multi-plasmid transient transfer method

The earliest rAAV production process used plasmids and helper viruses to infect packaging cells. At present, the three-plasmid co-transfection HEK 293 production process is mostly used. HEK293 cells contain the E1a and E1b genes of adenovirus (AdV), co-transfected with the transfer plasmid (containing the target gene and ITR sequence), and the structural plasmid ( Containing rep/cap gene) and helper plasmids (replicating viral genes E2A, E4, VARNA, etc.), rAAV can be recombined and packaged after 48-72 h.

This method is suitable for a variety of serotypes of rAAV, and the fermentation yield can generally reach 1014 V.G. per milliliter (vector genome), which can generally meet the amount of rAAV in early clinical trials (<1015 V.G.). However, due to the limitations of adherent culture methods, the multi-plasmid transient process is difficult to meet the needs of commercial production (>1016 V.G.).

2.1.2, stable production cell method

Construct a stable transfected HeLa cell line containing the helper gene rep/cap and the target gene, and the rAAV virus can also be packaged after adenovirus infection. Especially using the HeLa S3 cell line that can be cultured in suspension, the stable cell production method can be directly scaled up to a scale of 2 000 L. Similarly, the construction of BHK cells containing the ICP27 gene, after transfection with the replication-defective d27.1HSV containing the helper gene and the target gene, can also efficiently express rAAV.

However, the main disadvantages of the stable cell method are that cell construction and genetic stability studies are time-consuming, and the use of helper viruses in the production process poses a virus safety risk.

2.1.3 Baculovirus infection of insect cells method

Baculovirus has a high degree of species specificity, does not infect vertebrates, and can transfer rAAV genes and auxiliary function trans-acting elements to SF9 insect cells. Therefore, in recent years, it has also begun to be used in mass production of rAAV. The advantages of this method are that the baculovirus has good biological safety, high infection efficiency, and the production process is easy to scale up. However, the residual baculovirus and DNA-related substances should also be considered in quality control projects.

Key points of pharmacy evaluation:

For the upstream production process of rAAV, the pharmacy evaluation suggests that attention should be paid to the process development and verification of key processes. For example, the transient transfection of multiple plasmids or the addition of helper viruses should be used to explain the control range of key process parameters and the intermediate experience through process development and verification. Standards, such as the ratio of different vectors, the amount of transfection reagent, the multiplicity of infection of the helper virus and the production cell, etc.


2.2 Downstream process of AAV products

2.2.1, purification process

The rAAV purification process generally includes cell culture media harvesting, chemical/physical method lysis of cells, benzonase enzyme digestion to remove nucleic acid substances, multi-step chromatography and density gradient centrifugation, replacement of formulation formulation buffers and other processes. Among them, affinity chromatography simulates cells. The binding of the receptor captures rAAV. For example, heparin sulfate filler can specifically bind to rAAV2, and AVB agarose filler can bind to various serotypes of rAVV such as 1, 2, 3, 5 and 8 [22]. For the removal of empty carriers, iodixanol and cesium chloride ultracentrifugation methods are currently used.

It is worth noting that the relevant general review of the Chinese Pharmacopoeia clearly states that “unless otherwise proved to be reasonable, the cesium chloride-ethidium bromide density centrifugation method shall not be used for the purification of gene therapy products.”

2.2.2, virus removal verification

As mentioned above, baculovirus or helper virus (HSV, Ad5, etc.) are used when using baculovirus-insect cell or stable transfection cell line (HeLa) production process. Therefore, downstream processes should add specific virus removal/inactivation procedures.

For example: using surfactant TritonX-100 (0.5%, v/v) and adding a virus filtration process to remove residual baculovirus in the harvest liquid; using surfactants, low pH inactivation and other processes to remove HSV virus; using ions The exchange method, short-time heating (52 ℃, 10 min) and nanofiltration are used to remove adenovirus.

2.2.3, formulation prescription

Because rAAV capsid protein is not sensitive to temperature and pH changes, the formulation of rAAV products is relatively simple to develop. It is commonly used to add 200 mmol·L-1 magnesium sulfate or 0.001% poloxamer F68 to the salt solution to avoid the formation of aggregates, which can basically support long-term storage (-65 ℃) and transportation stability.

Evaluation points:

For the downstream production process of rAAV, the pharmacy evaluation recommends combining the removal efficiency of product-related impurities and process-related impurities to evaluate the rationality and robustness of the purification process. For the production process using the helper virus, the virus removal/inactivation process verification should be carried out in combination with the virus content in the harvest liquid, and the residual safety of the virus should be comprehensively evaluated.




3. Virus removal verification

3.1. Related impurities

3.1.1. Process-related impurities

The process-related impurities of rAAV products include host protein, host DNA, helper virus, plasmid, serum, and cesium chloride introduced in the virus packaging and purification process. Since virus packaging cells usually contain oncogenes, for example, HEK293 cells contain E1A adenovirus genes, and HeLa cells contain E6 and E7 oncogenes. Therefore, in general, the residual DNA content of the host cell should be less than 10 ng/Dose, and the residual DNA fragment should be less than 200 bp.

3.1.2. Product related impurities

Relevant impurities in rAAV products include empty viruses with unpackaged genes, viruses with incorrectly packaged genes (host DNA, incomplete target genes, helper virus genes, etc.), infective virus particles, aggregates or oxidized virus particles, etc.

These product-related impurities not only fail to achieve the expression of the target gene in target cells, but can also cause clinical immunogenicity or genotoxicity. For example, in a multi-plasmid sequence system, the proportion of empty viruses can reach 50% to 98%. Empty viruses are not capable of infecting, and are easy to form virus aggregates and degrade, causing an immune response in the body. Therefore, A260/A280, transmission electron microscopy, analytical centrifugation and mass spectrometry techniques should be used in quality research to detect empty virus content.

Replication-competent AAV (rcAAV) is due to the product-related impurities produced by the rAAV virus packaging rep and cap/AAP genes after homologous/non-homologous recombination occurs.
rcAAV can be replicated and amplified in the presence of helper virus. The detection of rcAAV generally adopts the use of sensitive cells for amplification in the presence of helper virus. After the cell lysate is amplified and passaged several times, Southern blot and qPCR are used to determine the rep or cap gene. For example, the rAAV product scAAV2/8-LP1-hFIXco, which has entered clinical trials, uses qPCR to control the content of rcAAV to less than 1/2.25×106. There are also reports that using optimized multi-plasmid transient technology, the limit of rcAAV content can be lower than 1/108 V.G.


3.2. General quality control items

The quality control of rAAV virus production includes process control and final product release testing (Table 2).

For example: The plasmid used for virus packaging should be subject to quality control such as identification, content, purity, host cell DNA residue, transfection efficiency, bacterial endotoxin, sterility, etc.;

The virus harvest liquid should control the detection of foreign factors (sterile, mycoplasma, etc.), foreign viruses, target viruses, etc.;

The release items of rAAV stock solution and preparation generally include appearance, physical and chemical properties (pH value, osmotic pressure), virus titer (physical titer, infectious titer), purity (protein purity, absorbance ratio, host DNA residue, plasmid DNA residue, nucleic acid) Enzyme residue), efficacy (target gene expression, in vitro activity, in vivo activity), safety
(Endotoxin, sterile, rcAAV).

In addition, for ophthalmic rAAV preparations, the endotoxin content should not be higher than 2.0 EU/dose/eye or 0.5 EU/mL, insoluble particles should be controlled according to ophthalmic preparations (USP<789>), and product release testing should include Products after configuration, etc.

3.3. Method for measuring potency

The method for determining the potency of rAAV products should reflect the physical titer, infectious activity and biological activity of the virus.

Generally, the qPCR method can be used to determine the viral genome in the early stage of clinical trials, and sensitive cells or targeted cells can be used to determine their infection ability and target product expression ability to characterize product efficacy.

After the key clinical trials are carried out, the in vitro enzyme activity method or the in vivo function experiment method that reflects the product’s mechanism of action should be further developed. For example, after SPK-RP65 enters Phase III clinical trials, the product efficacy is calculated by transfecting indicator cells (HEK293-LRAT) and measuring the retinol content of the protease-promoted product of RPE65.

It is worth pointing out that linear DNA should be used to draw a standard curve for the qPCR method of determining the viral genome. If supercoiled DNA is used as a reference, the measured value of the vector genome content is significantly higher than the true value.

IND/NDA pharmacy evaluation for gene therapy products application




4. Analysis of 3 examples of listed gene products

At present, only three rAAV gene therapy products (glybera, luxturna, and zolgensma) have been approved for marketing in the world. Most of these products in China are in the early stage of research and development, and the industry and regulators lack pharmaceutical research and evaluation of rAAV gene therapy products. Experience. The following is to discuss the key points and common problems of the pharmaceutical evaluation of such products based on the information disclosed in the evaluation reports of foreign marketed products.

4.1, Glybera

Glybera (alipogene tiparvovec, AAV1-LPLS447X) is an rAAV2 gene therapy product developed by Amsterdam Molecular Therapeutics that carries the human LPLS447X gene. It is clinically used to treat adult lipoprotein lipase deficiency by intramuscular injection.

There are major process changes during the process development of this product: the first-generation production process (AMT-010) uses the HEK293 expression system, and the commercial production process (ATM-011) uses the baculovirus-insect cell system expression, so pharmacy was developed. And non-clinical comparability studies.

In the downstream process, the process scale reduction model was used to verify the process of removing enveloped viruses and non-enveloped viruses.

The quality study conducted adequate characterization studies on 3 batches of continuous production batches, including: components (genomic integrity/size, protein analysis, molecular mass, capsid protein); physical properties (particle size, viral particle glycosyl) Chemical modification); primary structure (sequence confirmation, protein identification); advanced structure (lens electron microscopy, analytical ultracentrifugation); biological activity (infectious particles, specific activity, potency), etc.

The EMEA review concluded that because the cell harvest liquid contains a large amount of infectious baculovirus and the downstream process cannot provide sufficient virus removal effect, the applicant is required to provide a risk analysis report for clinical injection of baculovirus residual DNA, and it is recommended that the product be released Check items such as infectious baculovirus residue and rcAVV are added to the test items.

In addition, the EMEA review recommends that the production process after the product is on the market should further increase the baculovirus removal process (such as virus filtration), and the impurities (packaging cell DNA, residual Rep/Cap genes, rcAAV, infectious baculovirus, etc.) should be increased in the later stage. Sensitivity of detection method.


4.2 Luxturna

Luxturna (voretigene neparvovec-rzyl, SPK-RPE65) is a rAAV-2 gene therapy product carrying human RPE65 gene developed by Spark Therapeutics, USA. It is clinically used for subretinal injection to treat congenital amaurosis.

The product adopts three plasmids to co-transfect adherent HEK293 cells, and the production process of roller bottle. The production process includes: cell expansion, transfection, medium replacement, harvesting culture solution, tangential flow filtration, homogenization, ion exchange chromatography, density gradient centrifugation, preparation buffer replacement and filtration, etc.;

Before the phase III clinical trial of this product, the site, packaging container and other process changes occurred, and the “Side-by-side” method was used to conduct product comparability research on product quality.

Product release testing items include: physical and chemical (appearance, pH, concentration, extractable volume), identification (target gene), content (genome concentration), efficacy (target gene expression, in vitro activity), purity and safety items (endotoxin) , Particulate matter, sterile), etc. The host DNA, plasmid DNA, E1A gene and bovine serum albumin and other process-related impurities are controlled in quality research.

The FDA reviews that the stability of packaging cell HKE293 and the real-time stability of the product should continue to be completed after the product is marketed.

4.3, Zolgensma

Zolgensma (onasemnogene abeparvovec, AVXS-101) is a rAAV9 gene therapy product developed by AveXis that carries the Survival Motor Neuron (SMN) gene. It is clinically injected intravenously to treat spinal muscular atrophy in children.

In the process development process of this product, the risk assessment method is used to determine the key quality attributes of this product in combination with the target quality attributes, and the process scale reduction model is used to determine the process design space and process verification;

This product has undergone changes in site, process and formulations before the launch of the key clinical trials and before the registration and marketing.

The FDA reviewed and determined that the product purity and the effectiveness of the VG unit were consistent before and after the process change; the “potency” test items of this product include both quantitative detection of SMN protein expression levels in target cells, and in vivo semi-quantitative detection of mouse “survival rate”.

It is worth noting that due to the lack of precision and accuracy of the “content” analysis method of this product during the development of phase І clinical trials, an updated method was used to revise the clinical dosage after 44 months. In addition, in the stability experiment of this product, a decrease in “content” and “efficacy” was observed.





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