SC IV N. Gene Transfer Medicinal Products for Human Use
DEFINITION
For the purposes of this general chapter, gene transfer medicinal product (GTMP) shall mean a product obtained through a set of manufacturing processes aimed at the transfer, to be performed either in vivo or ex vivo, of a prophylactic, diagnostic or therapeutic gene (i.e. a piece of nucleic acid) to human/animal cells, and its subsequent expression in vivo. The gene transfer involves an expression system known as a vector, which can be of viral as well as non-viral origin. The vector can also be included in a human or animal cell.
Recombinant vectors, such as viral vectors and plasmids Recombinant vectors are either injected directly into the patient′s body (in vivo gene transfer) or transferred into host cells before administration of these genetically modified cells to the patient (ex vivo gene transfer). Viral vectors are derived from various viruses (for example, adenoviruses, poxviruses, retroviruses, lentiviruses, adeno-associated-viruses, herpesviruses). These vectors can be replicative, non-replicative or conditionally replicative. Plasmid vectors include nucleic acids in a simple formulation (for example, naked DNA) or complexed to various molecules (synthetic vectors such as lipids or polymers). Genetic material transferred by GTMPs consists of nucleotide sequences, which may notably encode gene products, antisense transcripts or ribozymes. Chemically synthesised oligonucleotides are not within the scope of this general chapter. After transfer, the genetic material may remain either cytoplasmic or episomal, or may be integrated into the host cell genome, depending on the integrating or non-integrating status of the vector.
Genetically modified cells Genetically modified eukaryotic or bacterial cells are modified by vectors to express a product of interest.
PRODUCTION
Substances used in production
The raw materials used during the manufacturing process, including viral seed lot and cell bank establishment, where applicable, are qualified. Unless otherwise justified, all substances used are produced within a recognised quality management system using suitable production facilities. Suitable specifications are established to control notably their identity, potency (where applicable), purity and safety in terms of microbiological quality and bacterial endotoxin contamination. The quality of water used complies with the relevant corresponding monographs (Purified water (0008), Highly purified water (1927), Water for injections (0169)). Where bovine serum is used, it complies with the monograph Bovine serum (2262). The use of antibiotics is avoided wherever possible during production.
Viral safety
The requirements of chapter 5.1.7 apply.
Transmissible spongiform encephalopathies (5.2.8)
A risk assessment of the product with respect to transmissible spongiform encephalopathies is carried out and suitable measures are taken to minimise such risk.
Recombinant vectors
PRODUCTION
For viral vectors, production is based on a cell bank system and a virus seed-lot system, wherever possible.
For plasmid vectors, production is based on a bacterial cell bank system.
The production method shall have been shown to yield a vector of consistent quality. Unless otherwise justified and authorised, the vector in the final product shall have undergone no more passages or subcultures from the master seed lot than were used to prepare the vector shown in clinical trials to be satisfactory with respect to safety and efficacy.
SUBSTRATE FOR VECTOR PROPAGATION
The substrates used comply with relevant requirements of the European Pharmacopoeia (5.2.2, 5.2.3, and the section Bacterial cells used for the manufacture of plasmid vectors for human use).
CHARACTERISATION OF THE VECTOR
Historical records of vector construction are documented, including the origin of the vector and its subsequent manipulation, notably deleted or modified regions.
The vector is characterised using suitable and validated methods.
The genetic stability of the vector at or beyond the maximum passage level or the maximum number of cell doublings of the cell line used for production is assessed by suitable methods.
PROPAGATION AND HARVEST
All processing of the cell banks and subsequent cell cultures is done in an area where no other cells or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media is qualified. The purity of the harvest is verified by suitable tests as defined in the corresponding specific sections.
PURIFIED HARVEST
The bulk of active substance is defined as a lot of purified recombinant vectors (viral vectors, or naked or complexed plasmids).
Unless otherwise justified and authorised, formulation and distribution of the final bulk is carried out under aseptic conditions using sterile containers (3.2).
The stability of the final lot is assessed using stability protocols including the duration, storage conditions, number of lots to be tested, test schedule and assays to be performed.
ASSAYS and TESTS
The GTMPs comply with assays and tests described in the corresponding specific sections.
Genetically modified cells
For cells to be modified with a recombinant vector, the data related to the recombinant vector are documented above, under Recombinant vectors.
PRODUCTION
CELL SUBSTRATE
For xenogeneic cell lines, including bacterial cells, a cell bank system comprising a master cell bank and working cell banks is established.
For autologous and allogeneic cells, a cell banking system comprising a master cell bank and working cell banks is established wherever possible.
TRANSFECTION / TRANSDUCTION
Cells are transfected or transduced using a recombinant vector (plasmid or viral vector) qualified as described under Recombinant vectors; the process is validated. They are handled under aseptic conditions in an area where no other cells or vectors are handled at the same time. All reagents used during cell manipulation steps are fully qualified. Antibiotics are avoided unless otherwise justified and authorised. Transfection or transduction is carried out under aseptic conditions.
In the case of frozen storage, the viability of genetically modified cells is assessed before freezing and after thawing.
If the cells are not used within a short period, stability is determined by verifying cell viability and expression of the genetic insert.
In the case of genetically modified cells encapsulated before implantation in man, any encapsulating component used is considered as part of the final product, and is therefore quality-controlled and fully characterised (for example, physical integrity, selective permeability, sterility).
ASSAYS and TESTS
Controls of xenogeneic, allogeneic or autologous cells include the following:
The competent authority may approve a reduced testing programme where necessary because of limited availability of cells. Where necessary because of time constraints, the product may be released for use before the completion of certain tests.
PLASMID VECTORS FOR HUMAN USE
DEFINITION
Plasmid vectors for human use are double-stranded circular forms of bacterial DNA that carry a gene of interest or a nucleotide sequence encoding antisense sequences or ribozymes and its expression cassette; they are amplified in bacteria extrachromosomally. They are used to transfer genetic material into human somatic cells in vivo or to genetically modify autologous, allogeneic, xenogeneic or bacterial cells before administration to humans. Plasmid vectors may be presented as naked DNA or may be formulated with synthetic delivery systems such as lipids (lipoplexes), polymers (polyplexes) and/or peptide ligands that facilitate transfer across the cell membrane and delivery to the cell, or that target delivery via specific receptors.
Plasmids formulated with synthetic delivery systems are not within the scope of this section.
PRODUCTION
Plasmid construction
A typical plasmid vector is composed of:
A complete description of the plasmid DNA, including its nucleotide sequence, is established with the identification, source, means of isolation and nucleotide sequence of the genetic insert. The source and function of component parts of the plasmid, such as the origin of replication, viral and eukaryotic promoters and genes encoding selection markers, are documented.
Cell banks
Production of plasmid vectors is based on a bacterial cell-bank system with generation and characterisation of a master cell bank (MCB), working cell banks (WCBs) and end-of-production cells (EOPCs), which comply with the section Bacterial cells used for the manufacture of plasmid vectors for human use. The raw materials used during the manufacturing process, including cell bank establishment, are qualified.
Selection techniques
Unless otherwise justified and authorised, antibiotic-resistance genes used as selectable genetic markers, particularly for clinically useful antibiotics, are not included in the vector construct. Other selection techniques for the recombinant plasmid are preferred.
Reference standards
A suitable batch of the formulated plasmid, preferably one that has been clinically evaluated, is fully characterised and retained for use as a reference standard as necessary in routine control tests.
PROPAGATION AND HARVEST
Plasmid DNA is transferred to host strain bacterial cells and a single clone of transformed bacteria is expanded to create the MCB. The WCB is then derived from the MCB. The EOPCs are obtained from the WCB by fermentation in production conditions.
Plasmid DNA is isolated from harvested cells using an extraction step and is purified to obtain the bulk product.
Unless otherwise justified and authorised, caesium chloride-ethidium bromide density gradients are not used for production.
Purified plasmid
The production process is optimised to remove impurities consistently while retaining product activity. The requirement to test for a particular impurity depends on the following:
If selective resistance to specific antibiotics has been used for selection, data from validation studies of purification procedures are required to demonstrate the clearance capability for residual antibiotics.
Relevant in-process controls are performed to ensure that the process is continuously under control, for example, amount and form of plasmid after the extraction steps and amount of endotoxins after the extraction steps.
Only a batch of purified plasmid that complies with the following requirements may be used.
Identity and integrity of the purified plasmid
Identity and integrity of the purified plasmid are established by suitable methods such as sequencing or nucleic acid amplification techniques (NAT) (2.6.21; restriction enzyme analysis may be used where it is sufficient to detect potential critical modifications in the plasmid and confirm the plasmid identity.
Plasmid DNA
The following indications are given as examples.
DNA concentrations greater than 500 ng/mL may be determined using absorbance measurement at 260 nm. A 50 µg/mL double-stranded DNA solution has an absorbance of 1 (specific absorbance 200).
DNA concentrations less than 500 ng/mL are determined following incubation with fluorescent dyes that bind specifically to double-stranded DNA, using a reference standard of DNA to establish a calibration curve.
Liquid chromatography may also be used to determine the concentration of plasmid DNA using a reference standard. In some cases, capillary electrophoresis is also acceptable.
DNA forms
Plasmid DNA is characterised in terms of the proportions of supercoiled, multimeric, relaxed monomer and linear forms, using suitable analytical methods, examples of which are given below. For quantification of supercoiled forms, anion-exchange high performance liquid chromatography (HPLC) or capillary electrophoresis may be used. Capillary electrophoresis is also suitable for the quantification of other forms.
Residual host-cell DNA
The content of residual host-cell DNA is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Residual RNA
The content of residual RNA is determined, unless the process has been validated to demonstrate suitable clearance. Reverse-phase HPLC (RP-HPLC) may be used, or quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) (2.6.21) when a lower limit of detection is required.
Residual host-cell protein
The concentration of residual host-cell protein is determined using standard protein assays (2.5.33), SDS-PAGE followed by silver staining, or specific immuno-assays such as western blot or ELISA, unless the process has been validated to demonstrate suitable clearance.
Microbiological control
Depending on the preparation concerned, it complies with the test for sterility (2.6.1) or the bioburden is determined (2.6.12).
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
final bulk
Several purified harvests may be pooled during preparation of the final bulk. A stabiliser and other excipients may be added. The formulated product is filtered through a bacteria-retentive filter.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.
Sterility (2.6.1)
It complies with the test for sterility.
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.
IDENTIFICATION
The plasmid vector is identified by restriction enzyme analysis or by sequencing. The test for biological activity also serves to identify the product.
TESTS
Tests carried out on the final lot include the following.
Appearance
pH (2.2.3)
Within the limits approved for the particular preparation.
Extractable volume (2.9.17)
It complies with the test for extractable volume.
Residual moisture (2.5.12)
Within the limits approved for the particular freeze-dried preparation.
DNA forms
The percentage of the specific monomeric supercoiled form is determined as described for the purified plasmid.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
ASSAY
Plasmid DNA
Not less than the quantity stated on the label, determined, for example, by 1 of the following methods.
DNA concentrations greater than 500 ng/mL may be determined using absorbance measurement at 260 nm. A 50 µg/mL double-stranded DNA solution has an absorbance of 1 (specific absorbance 200).
DNA concentrations less than 500 ng/mL are determined following incubation with fluorescent dyes that bind specifically to double-stranded DNA, using a reference standard of DNA to establish a calibration curve.
Liquid chromatography may also be used to determine the concentration of plasmid DNA using a reference standard. In some cases, capillary electrophoresis is also acceptable.
Biological activity
Wherever possible, biological activity is assessed through in vitro or in vivo bioassays. A well-defined, representative reference standard is required as a positive control for the assay. Bioassays employed to assay plasmid vectors generally involve transfection of a relevant cell line in vitro, followed by some functional measure of the expressed genetic insert. Such functional assays provide information about the activity of the product encoded by the genetic insert instead of the expression level of the genetic insert itself.
It may be necessary to supplement the bioassay with western-blot and ELISA assays to assess the integrity and quantity of the expressed product.
LABELLING
BACTERIAL CELLS USED FOR THE MANUFACTURE OF PLASMID VECTORS FOR HUMAN USE
Production of plasmid vectors for human use is based on the use of a bacterial cell-bank system with generation and characterisation of a master cell bank (MCB), working cell banks (WCBs) and end-of-production cells (EOPCs). A bacterial cell bank for the manufacture of plasmid vectors is a collection of vials containing bacterial cells stored under defined conditions, with uniform composition, and obtained from pooled cells derived from a single clone of a transformed host strain. The MCB has a known, documented history; it is preferably derived from a qualified repository source. The WCB is produced by expanding one or more vials of the MCB. Methods and reagents used to produce the bank and storage conditions are documented.
MCBs and WCBs are qualified by testing an aliquot of the banked material or testing a subculture of the cell bank.
The following table indicates the tests required at each stage of production.
IDENTITY AND PURITY TESTING
Viability
The number of viable cells is determined by plating a diluted aliquot of bacterial cells on an appropriate medium and counting individual colonies.
Biochemical and physiological bacterial strain characterisation
Depending on the bacterial strain used for production, relevant biochemical and physiological characterisation is performed to confirm cell identity at the species level.
Genotyping / phenotyping
The genotype of bacterial cells is verified by determination of the suitable specific phenotypic markers or by appropriate genetic analysis.
Presence of the plasmid
Sequencing The whole nucleotide sequence of the plasmid is verified.
Copy number The plasmid DNA is isolated and purified from a known number of bacteria and the copy number determined by a suitable method such as quantitative PCR (2.6.21).
Restriction map Restriction endonuclease digestion is performed with sufficient resolution to verify that the structure of the plasmid is unaltered in bacterial cells.
Percentage of cells retaining the plasmid Bacterial elements present in the plasmid, such as selectable genetic markers, are used to define the percentage of bacteria retaining the plasmid.
ADVENTITIOUS AGENTS AND ENDOGENOUS VIRUSES
Purity by plating
Bacterial cells are streaked out onto suitable media and incubated in the required conditions in order to detect potential bacterial contaminants. In order to test for inhibition of the growth of contaminating organisms, additional tests in the presence of a definite amount of relevant positive control bacteria are carried out. A suitable number of colonies is examined; no contamination is detected.
Presence of bacteriophage
Bacterial cells are plated and incubated in a medium allowing proliferation of bacteriophages, to test for bacteriophage presence. The test is validated by the use of a reference bacteriophage strain and permissive cells as positive controls. A suitable number of colonies is examined; no contamination is detected.
ADENOVIRUS VECTORS FOR HUMAN USE
DEFINITION
Adenovirus vectors for human use are freeze-dried or liquid preparations of recombinant adenoviruses, genetically modified to transfer genetic material to human somatic cells in vivo or ex vivo.
PRODUCTION
VECTOR CONSTRUCTION
There are different approaches for the design and construction of an adenovirus vector. The purpose of clinical use determines which approach is optimal. A method is chosen that minimises the risk of generating replication-competent adenovirus vectors or that effectively eliminates helper viruses that might be used during production.
VECTOR PRODUCTION
The production method shall have been shown to yield a vector of consistent quality. Unless otherwise justified and authorised, the vector in the final product shall have undergone no more passages from the master seed lot than were used to prepare the vector shown in clinical trials to be satisfactory with respect to safety and efficacy.
The genetic and phenotypic stability of the vector at or beyond the maximum passage level used for production is assessed by suitable methods.
SUBSTRATE FOR Vector PROPAGATION
The vector is propagated in continuous cell lines (5.2.3) based on a cell bank system. The occurrence of replication-competent adenoviruses may be significant when large regions of homology exist between the viral genome and the genome of the complementation cells. This occurrence may be minimised by minimising the homology between both genomes. The use of cells with no sequence homology with the vector is recommended for production.
VECTOR SEED LOT
Production of the vector is based on a seed-lot system.
The strain of adenovirus used is identified by historical records that include information on its origin and its subsequent manipulation, notably deleted or modified regions. A detailed description of the genetic insert(s) and the flanking control regions is established, including the nucleotide sequence. The method by which the genetic insert is introduced into the vector is documented.
Only a seed lot that complies with the following requirements may be used for vector production.
Identification
The vector is identified in the master seed lot and each working seed lot by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Genetic and phenotypic characterisation
The following tests are carried out.
Vector concentration
The titre of infectious vector or the concentration of vector particles in the master seed lot and each working seed lot are determined.
Extraneous agents (2.6.16)
The master seed lot and each working seed lot comply with the tests for extraneous agents.
Replication-competent adenoviruses
Replication-competent adenoviruses are generated by homologous recombination between the recombinant viral DNA and the adenovirus sequences integrated into the genome of the complementation cells.
Detection of replication-competent adenoviruses is performed by a suitable method approved by the competent authority. It is generally performed by an infectivity assay on sensitive detector cell lines, which are not able to complement for the genes deleted from the vector. Other indicators of viral replication may be used as appropriate.
When replication-competent adenoviruses are not supposed to be present in the test sample, considering vector construction and cell lines used, at least 2, but preferably 3 or 4 successive passages are performed on the detector cell line, where applicable. Detection of a cytopathic effect at the end of the passages reveals the presence of replication-competent adenoviruses in the preparation. Positive controls are included in each assay to monitor its sensitivity.
When replication-competent adenoviruses are expected to be present in the test sample, plaque-assays or limit dilution assays on a detector cell line may be performed.
PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is done in an area with a suitable containment level where no other cells or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media is qualified. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration, but it is preferable to have a substrate free from antibiotics during production. Unless otherwise justified and authorised, at no stage during production is penicillin or streptomycin used. A portion of the production cell cultures is set aside as uninfected cell cultures (control cells).
Each single harvest that complies with the following requirements may be used in the preparation of the purified harvest.
Identification
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Vector concentration
The titre of infectious vector and the concentration of vector particles in single harvests are determined.
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous agents.
Control cells
Control cells comply with a test for identification (5.2.3) and a test for extraneous agents (2.6.16).
PURIFIED HARVEST
Several single harvests may be pooled before the purification process. The purification process is validated to demonstrate the satisfactory removal of impurities.
Purified harvests that comply with the following requirements may be used in the preparation of the final bulk.
Identification
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Genomic integrity
Genomic integrity of the vector is verified by suitable methods such as restriction enzyme analysis.
Vector concentration
The titre of infectious vector and the concentration of vector particles in purified harvests are determined.
Residual host-cell protein
The concentration of residual host-cell protein is determined by a suitable immunochemical method (2.7.1), unless the process has been validated to demonstrate suitable clearance.
Residual host-cell DNA
The content of residual host-cell DNA is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative polymerase chain reaction (PCR) is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Residual reagents
Where reagents are used during the production process, tests for these substances are carried out on the purified harvest, unless the process has been validated to demonstrate suitable clearance.
Residual antibiotics
Where antibiotics are used during the production process, their residual concentration is determined by a microbiological assay (adapted from general method 2.7.2) or by other suitable methods (for example, liquid chromatography), unless the process has been validated to demonstrate suitable clearance.
FINAL BULK
Several purified harvests may be pooled during preparation of the final bulk. A stabiliser and other excipients may be added. The formulated product is filtered through a bacteria-retentive filter.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.
Sterility (2.6.1)
It complies with the test for sterility.
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.
Provided that the tests for bovine serum albumin (when bovine serum is used to manufacture the vector) and replication-competent adenoviruses have been carried out with satisfactory results on the final bulk, they may be omitted on the final lot.
IDENTIFICATION
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
TESTS
Osmolality (2.2.35)
Within the limits approved for the particular preparation.
pH (2.2.3)
Within the limits approved for the particular preparation.
Extractable volume (2.9.17)
It complies with the test for extractable volume.
Residual moisture (2.5.12)
Within the limits approved for the particular freeze-dried preparation.
Bovine serum albumin
Not more than the limit approved for the particular preparation, determined by a suitable immunochemical method (2.7.1), where bovine serum has been used during production.
Replication-competent adenovirus concentration
Within the limits approved for the particular preparation.
Vector aggregates
Vector aggregates are determined by suitable methods (for example, light scattering).
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
Thermal stability
Maintain samples of the vector final lot at a temperature and for a length of time that are adapted and authorised for the particular preparation. Determine the total infectious vector concentration after heating, as described below under Assay. Determine in parallel the vector concentration of a non-heated sample. The estimation of the difference between the total vector concentration without heating and after heating is within the limits approved for the particular preparation.
ASSAY
Vector particle concentration
Physical titration is performed by a suitable technique (for example, liquid chromatography, absorbance measurement or NAT (2.6.21)). Use an appropriate vector reference standard to validate each assay.
The vector particle concentration of the preparation to be examined is not less than the concentration stated on the label.
Infectious vector titre
Titrate the preparation to be examined by inoculation into cell cultures. Titrate an appropriate vector reference standard to validate each assay.
The assay is invalid if:
Ratio of vector particle concentration to infectious vector titre
Within the limits approved for the particular preparation.
Expression of the genetic insert product
The expression of the genetic insert product(s) is determined wherever possible, following inoculation of cell cultures with the particular preparation at a predetermined multiplicity of infection, by suitable immunochemical (2.7.1) or biochemical assays or by flow cytometry (2.7.24).
Biological activity
Unless otherwise justified and authorised, biological activity is determined by a suitable in vitro or in vivo test.
LABELLING
POXVIRUS VECTORS FOR HUMAN USE
DEFINITION
Poxvirus vectors for human use are freeze-dried or liquid preparations of recombinant poxviruses, genetically modified to transfer genetic material to human somatic cells in vivo or ex vivo.
PRODUCTION
VECTOR CONSTRUCTION
The general design of a poxvirus vector is currently as follows: the genetic insert is inserted downstream of a poxvirus promoter. This expression cassette is inserted into the poxvirus genome in such a manner that it interrupts a viral gene non-essential for replication or is positioned between 2 virus open reading frames.
In most strategies used so far for the construction of the vector, the expression cassette is first inserted within the target site of a virus DNA fragment cloned into a bacterial plasmid. The plasmid is then introduced into host cells, cultured in vitro, which are simultaneously infected with the parental poxvirus. DNA recombination occurs within the infected cells, between homologous sequences in the viral genome and viral sequences in the plasmid so as to transfer the genetic insert into the targeted site of the viral genome. The correct targeting of the inserted DNA is checked by restriction-enzyme mapping, NAT (2.6.21) and sequencing. Successive plaque-cloning steps are performed to purify the recombinant poxvirus from the mixture of parental and recombinant poxviruses. A variety of methods (for example, foreign marker genes, DNA hybridisation, immunological detection, phenotypic changes in the virus) are employed to facilitate recognition and/or selection of the recombinant poxvirus from the background of parental virus. Where foreign marker genes have been transiently employed, they are removed by appropriate methods from the final recombinant poxvirus.
An alternative strategy for creating poxvirus vectors begins with the in vitro construction of a full-length virus genome harbouring the expression cassette within a chosen target site. This recombinant genome is then introduced into host cells simultaneously infected with a helper poxvirus that is unable to multiply. The helper virus may be a poxvirus of the same species whose ability to multiply has been inactivated, or another poxvirus species that does not multiply in the host cells.
The construction of non-replicative poxvirus vectors relies on specific host cell lines or primary cells that are naturally permissive, or on host cell lines that have been modified to express an essential poxvirus gene. These cells fulfill the general requirements for the production of medicinal products (5.2.3) and do not allow the generation of replicative vectors.
VECTOR PRODUCTION
The production method shall have been shown to yield a vector of consistent quality. Unless otherwise justified and authorised, the vector in the final product shall have undergone no more passages from the master seed lot than were used to prepare the vector shown in clinical trials to be satisfactory with respect to safety and efficacy.
The genetic and phenotypic stability of the vector at or beyond the maximum passage level used for production is assessed by suitable methods.
SUBSTRATE FOR VECTOR PROPAGATION
The vector is propagated under aseptic conditions in human diploid cells (5.2.3), in continuous cell lines (5.2.3) or in cultures of chick-embryo cells derived from a chicken flock free from specified pathogens (5.2.2). When the vector is propagated in a continuous cell line or in human diploid cells, a cell-bank system is established.
VECTOR SEED LOT
Production of the vector is based on a seed-lot system.
The strain of poxvirus used is identified by historical records that include information on its origin and its subsequent manipulation, notably deleted or modified regions. A detailed description of the genetic insert(s) and the flanking control regions is established, including the nucleotide sequence. The method by which the genetic insert is introduced into the vector is documented.
Only a seed lot that complies with the following requirements may be used for vector production.
Identification
The vector is identified in the master seed lot and each working seed lot by immunochemical methods (2.7.1) or NAT (2.6.21).
Genetic and phenotypic characterisation
The following tests are carried out.
Infectious vector titre
The titre of infectious vector in the master seed lot and each working seed lot is determined.
Extraneous agents (2.6.16)
The master seed lot and each working seed lot comply with the tests for extraneous agents, except where cytopathic strains cannot be neutralised and the vector causes interference. Where a test cannot be performed, carry out a suitable validated alternative.
PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area with a suitable containment level where no other cells or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media is qualified. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration, but it is preferable to have a substrate free from antibiotics during production. Unless otherwise justified and authorised, at no stage during production is penicillin or streptomycin used. A portion of the production cell culture is set aside as uninfected cell cultures (control cells).
Each single harvest that complies with the following requirements may be used in the preparation of the purified harvest.
Identification
The vector is identified by immunochemical methods (2.7.1) or NAT (2.6.21).
Infectious vector titre
The titre of infectious vector in single harvests is determined.
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous agents, except where cytopathic strains cannot be neutralised and the vector causes interference. Where a test cannot be performed, carry out a suitable validated alternative.
Control cells
If human diploid cells or a continuous cell line are used for production, the control cells comply with a test for identification (5.2.3). They comply with the tests for extraneous agents (2.6.16).
PURIFIED HARVEST
Processing is carried out under aseptic conditions. Several single harvests may be pooled before the purification process. The harvest is first clarified to remove cells and then, where applicable, purified by validated methods.
Purified harvests that comply with the following requirements may be used in the preparation of the final bulk.
Identification
The vector is identified by immunochemical methods (2.7.1) or NAT (2.6.21).
Genomic integrity
Genomic integrity of the vector is verified by suitable methods such as restriction enzyme analysis.
Infectious vector titre
The titre of infectious vector in purified harvests is determined.
Ratio of infectious vector titre to total protein concentration
The total protein concentration is determined by a suitable method (2.5.33). The ratio between infectious vector titre and total protein concentration is calculated.
Residual host-cell protein
The concentration of residual host-cell protein is determined by a suitable immunochemical method (2.7.1), unless the process has been validated to demonstrate suitable clearance.
Residual host-cell DNA
The content of residual host-cell DNA is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Residual reagents
Where reagents are used during the production process, tests for these substances are carried out on the purified harvest, unless the process has been validated to demonstrate suitable clearance.
Residual antibiotics
Where antibiotics are used during the production process, their residual concentration is determined by a microbiological assay (adapted from general method 2.7.2) or by other suitable methods (for example, liquid chromatography), unless the process has been validated to demonstrate suitable clearance.
FINAL BULK
Several purified harvests may be pooled during preparation of the final bulk. A stabiliser and other excipients may be added.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.
Sterility (2.6.1)
It complies with the test for sterility.
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.
Provided that the test for bovine serum albumin (when bovine serum is used to manufacture the vector) has been carried out with satisfactory results on the final bulk, it may be omitted on the final lot.
IDENTIFICATION
The vector is identified by immunochemical methods (2.7.1) or NAT (2.6.21).
TESTS
Osmolality (2.2.35)
Within the limits approved for the particular preparation.
pH (2.2.3)
Within the limits approved for the particular preparation.
Extractable volume (2.9.17)
It complies with the test for extractable volume.
Residual moisture (2.5.12)
Within the limits approved for the particular freeze-dried preparation.
Bovine serum albumin
Not more than the limit approved for the particular preparation, determined by a suitable immunochemical method (2.7.1), where bovine serum has been used during production.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
Thermal stability
Maintain samples of the vector final lot at a temperature and for a length of time that are adapted and authorised for the particular preparation. Determine the total infectious vector concentration after heating, as described below under Assay. Determine in parallel the vector concentration of a non-heated sample. The estimation of the difference between the total vector concentration without heating and after heating is within the limits approved for the particular preparation.
ASSAY
Infectious vector titre
Titrate at least 3 vials of the preparation to be examined by inoculation into cell cultures. Titrate a vial of an appropriate vector reference standard to validate each assay.
The vector titre of the preparation to be examined is not less than the minimum titre stated on the label.
The assay is invalid if:
Expression of the genetic insert product
The expression of the genetic insert product(s) is determined, wherever possible, following inoculation of cell cultures with the particular preparation at a predetermined multiplicity of infection, by suitable immunochemical (2.7.1) or biochemical assays or by flow cytometry (2.7.24).
Biological activity
Unless otherwise justifed and authorised, biological activity is determined by a suitable in vitro or in vivo test.
LABELLING
RETROVIRIDAE-DERIVED VECTORS FOR HUMAN USE
DEFINITION
Retroviridae-derived vectors for human use are liquid or freeze-dried preparations of recombinant retroviruses, lentiviruses or spumaviruses, genetically modified to render them replication-incompetent, which are used to transfer genetic material to human somatic cells in vivo or ex vivo. This section applies to non-replicative vectors.
PRODUCTION
VECTOR CONSTRUCTION
A typical vector is composed of:
The vector construction is designed to prevent the generation of replication-competent viruses.
VECTOR PRODUCTION
The production method shall have been shown to yield a vector of consistent quality. Unless otherwise justified and authorised, the packaging or producer cells shall have undergone no more cell doublings from the master cell bank (MCB) than were used to prepare the vector shown in clinical trials to be satisfactory with respect to safety and efficacy.
The genetic and phenotypic stability of the packaging or producer cells at or beyond the maximum number of cell doublings used for production is assessed by suitable methods.
Vectors are produced in continuous cell lines (5.2.3) using a cell-bank system. Production may involve either stably or transiently transfected cells.
DEFINITIONS
Packaging cells A source cell line stably transfected with plasmids containing the viral genes necessary for production of empty vector particles: gag, pol, env.
Producer cells Contain the viral genes and expression cassette necessary for vector production.
PRODUCTION INTERMEDIATES
Packaging cells
Copy number
The genomic DNA is isolated and purified from a known number of cells and the gag, pol and env genes copy number is determined by a suitable method such as quantitative PCR (2.6.21).
Sequence integrity of the viral genes
Complete nucleotide sequencing of the inserted viral genes and their regulatory elements is performed.
Genetic stability
Genetic stability of the packaging cells is verified at or beyond the maximum number of cell doublings used for production.
Plasmids
Production of the vector requires the use of plasmid intermediates. For each plasmid DNA used during production, a complete description is established, including identification, source, means of isolation and nucleotide sequence. The source and function of component parts of these plasmids, such as the origin of replication, viral and eukaryotic promoters and genes encoding selection markers, are documented.
Production of plasmid intermediates is based on a bacterial cell-bank system. The MCB complies with the requirements of the section Bacterial cells used for the manufacture of plasmid vectors for human use. Plasmids are purified by suitable techniques.
Only plasmid batches that comply with the following requirements may be used for the production of the vector.
Identification
Plasmids are identified by restriction enzyme analysis, sequencing or NAT (2.6.21).
Genomic integrity
Genomic integrity of the plasmid is verified by suitable methods such as restriction enzyme analysis of the viral genes, the genetic insert and their respective regulation elements.
Plasmid DNA
The following indications are given as examples.
DNA concentrations greater than 500 ng/mL may be determined using absorbance measurement at 260 nm. A 50 µg/mL double-stranded DNA solution has an absorbance of 1 (specific absorbance 200).
DNA concentrations less than 500 ng/mL are determined following incubation with fluorescent dyes that bind specifically to double-stranded DNA, using a reference standard of DNA to establish a calibration curve.
Liquid chromatography may also be used to determine the concentration of plasmid DNA using a reference standard. In some cases, capillary electrophoresis is also acceptable.
Residual host-cell DNA
The content of residual host-cell DNA is determined using a suitable method, unless the production process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
Sterility (2.6.1)
It complies with the test for sterility.
Producer cells used in a stable production system
Copy number
The copy number of the integrated viral genes and expression cassette is determined by a suitable method.
Genetic stability
Genetic stability of the producer cells at or beyond the maximum number of cell doublings used for production is confirmed.
Sequence integrity of the viral genes and expression cassette
Complete nucleotide sequencing of the inserted viral genes, the expression cassette and their respective regulation elements (for example, LTRs, promoters, psi sequence, polyadenylation signal) is performed.
Replication-competent viruses
The detection of replication-competent viruses is performed by suitable methods. Detection may be based on a co-cultivation for several cell doublings of the producer cells with a permissive cell line, followed by detection (either by observation of a cytopathic or haemadsorbing effect on indicator cells like PG4 S+L-, by detection using indicator cell lines by NAT (2.6.21) or by marker-rescue assay). Positive controls are included in each assay to monitor its sensitivity. No replication competent viruses are found.
PRODUCTION AND HARVEST
All processing of the cell bank and subsequent cell cultures is done in an area with a suitable containment level where no other cells or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media must be qualified. It is preferable to have a substrate free from antibiotics during production. Unless otherwise justified and authorised, at no stage during production is penicillin or streptomycin used.
Each single harvest that complies with the following requirements may be used in the preparation of the purified harvest.
Identification
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Vector concentration
The titre of infectious vector and/or the concentration of vector particles in single harvests is determined.
Extraneous agents
Each single harvest complies with the tests for extraneous agents (2.6.16).
Control cells
Where a transient production process is used, control cells comply with a test for identification (5.2.3) and a test for extraneous agents (2.6.16).
PURIFIED HARVEST
Several single harvests may be pooled before purification. Purified harvests that comply with the following requirements may be used in the preparation of the final bulk.
Identification
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Genomic integrity
Genomic integrity of the vector is verified by a suitable method.
Vector concentration
The infectious particle titre is determined by a suitable method, for example infection of permissive cells followed by quantitative NAT (for example, quantitative PCR), Southern blot or protein expression. For lentivirus vectors, the physical titre is measured, for example by ELISA (p24).
Replication-competent viruses
Detection of replication-competent viruses is performed by suitable methods. It is generally performed by amplification on permissive cells followed by NAT (2.6.21), by detection of a viral antigen (for example, p24 by ELISA) or by marker-rescue assay. Positive controls are included in each assay to monitor its sensitivity.
Detection of replication-competent viruses is performed on the purified harvest or on the final lot. No replication-competent viruses are found.
Residual host-cell protein
The concentration of residual host-cell protein is determined by a suitable immunochemical method (2.7.1), unless the process has been validated to demonstrate suitable clearance.
Residual host-cell DNA
The content of residual host-cell DNA is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Residual reagents
Where reagents are used during production, tests for these substances are carried out on the purified harvest, unless the process has been validated to demonstrate suitable clearance.
Residual antibiotics
Where antibiotics are used during the production process, their residual concentration is determined by a microbiological assay (adapted from general method 2.7.2) or by other suitable methods (for example, liquid chromatography), unless the process has been validated to demonstrate suitable clearance.
Residual plasmids
Where a transient production process is used, the concentration of residual contaminating plasmids must be quantified.
FINAL BULK
Several purified harvests may be pooled during preparation of the final bulk. A stabiliser and other excipients may be added. The formulated product is filtered through a bacteria-retentive filter.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.
Sterility (2.6.1)
It complies with the test for sterility.
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.
Provided that the tests for bovine serum albumin (when bovine serum is used to manufacture the vector) and replication-competent viruses have been carried out with satisfactory results on the purified harvest, they may be omitted on the final lot.
IDENTIFICATION
Retroviridae-derived vectors are identified by NAT (2.6.21), immunochemical methods (2.7.1) or restriction enzyme analysis.
TESTS
Osmolality (2.2.35)
Within the limits approved for the particular preparation.
pH (2.2.3)
Within the limits approved for the particular preparation.
Extractable volume (2.9.17)
It complies with the test for extractable volume.
Residual moisture (2.5.12)
Within the limits approved for the particular freeze-dried preparation.
Bovine serum albumin
Where bovine serum has been used during production, not more than the limit approved for the particular preparation, determined by a suitable immunochemical method (2.7.1).
Replication-competent viruses
Detection of replication-competent viruses is performed by suitable methods. It is generally performed by amplification on permissive cells followed by NAT (2.6.21), detection of a viral antigen (for example, p24 by ELISA) or marker-rescue assay. Positive controls are included in each assay to monitor its sensitivity.
Detection of replication-competent viruses is performed on the purified harvest or on the final lot. No replication-competent viruses are found.
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
ASSAY
Vector-particle concentration
Physical titration is performed by a suitable technique (for example, immunochemical methods (2.7.1) or NAT (2.6.21)). Use an appropriate vector reference standard to validate each assay.
Infectious vector titre
Titrate the preparation to be examined by inoculation into cell cultures. Titrate an appropriate vector reference standard to validate each assay.
The infectious vector titre of the preparation to be examined is not less than the minimum titre stated on the label.
The assay is invalid if:
Ratio of vector-particle concentration to infectious vector titre
Within the limits approved for the particular product, where applicable.
Expression of the genetic insert product
The expression of the genetic insert product(s) is determined wherever possible, following inoculation of cell cultures with the product at a predetermined multiplicity of infection, by suitable immunochemical (2.7.1) or biochemical assays or by flow cytometry (2.7.24).
Biological activity
Unless otherwise justified and authorised, biological activity is determined by a suitable in vitro or in vivo test.
LABELLING
ADENO-ASSOCIATED-VIRUS VECTORS FOR HUMAN USE
DEFINITION
Adeno-associated-virus (AAV) vectors for human use are freeze-dried or liquid preparations of recombinant AAV (rAAV), genetically modified to transfer genetic material to human somatic cells in vivo or ex vivo.
PRODUCTION
VECTOR CONSTRUCTION
rAAV vectors are developed by replacement of the rep and cap genes with the genetic insert of interest. The inverted terminal repeat (ITR) sequences are retained in the rAAV vector since these are the only AAV sequences absolutely required in cis to function as the origin of replication. The rep and cap genes are required in trans and function for replication and packaging respectively. In summary, the rAAV vector contains the ITRs and the genetic insert.
Wild-type AAV normally replicate only in the presence of helper functions, provided by a coinfecting adenovirus or herpes virus. Therefore, there are different approaches to the manufacture of an AAV vector. The manufacturing strategy chosen is designed to minimise the risk for the generation of replication-competent AAV vectors and effectively eliminate helper viruses that might be used during production.
VECTOR PRODUCTION
The production method shall have been shown to yield a vector of consistent quality and stability.
To produce AAV vectors, several strategies are currently used, for example:
Depending on the strategy used to produce AAV vectors, different production intermediates are required (plasmids, viruses used for production, packaging cells).
The occurrence of replication-competent AAV may be significant when regions of homology exist between the genomes of the production intermediates and the rAAV vector. This occurrence may be minimised by reducing the homology between these genomes to a minimum. The use of production intermediates with no sequence homology is recommended for production.
The genetic and phenotypic stability of the vector at or beyond the maximum number of passage levels used for production is assessed by suitable methods.
PRODUCTION INTERMEDIATES
Viruses used for production and the rAAV vector are produced in continuous cell lines (5.2.3) using a seed lot and a cell-bank system.
Packaging and producer cells
Copy number
The genomic DNA is isolated and purified from a known number of cells and the copy number of the inserted viral genes and of the expression cassette is determined by a suitable method such as quantitative PCR (2.6.21).
Sequence integrity of the viral genes and expression cassette
Complete nucleotide sequencing of the inserted viral genes, of their regulatory elements and where applicable, of the expression cassette is performed.
Genetic stability
Genetic stability of the cells is verified at or beyond the maximum number of cell doublings used for production.
Wild-type AAV
The absence of wild-type AAV is verified using NAT (2.6.21).
Plasmids
Production of the AAV vector by transient co-transfection requires the use of plasmid intermediates. For each plasmid DNA used during production, a complete description is established, including identification, source, means of isolation and nucleotide sequence. The source and function of component parts of these plasmids, such as the origin of replication, viral and eukaryotic promoters and genes encoding selection markers, are documented.
Production of plasmid intermediates is based on a bacterial cell-bank system. The master cell bank complies with the requirements of the section Bacterial cells used for the manufacture of plasmid vectors for human use. Plasmids are purified by suitable techniques.
Only plasmid batches that comply with the following requirements may be used for the production of the AAV vector.
Identification
Plasmids are identified by restriction enzyme analysis, sequencing or NAT (2.6.21).
Genomic integrity
Genomic integrity of the plasmid is verified by suitable methods such as restriction enzyme analysis of the region corresponding to rep, cap and the expression cassette.
Plasmid DNA
The following indications are given as examples.
DNA concentrations greater than 500 ng/mL may be determined using absorbance measurement at 260 nm. A 50 µg/mL double-stranded DNA solution has an absorbance of 1 (specific absorbance 200).
DNA concentrations less than 500 ng/mL are determined following incubation with fluorescent dyes that bind specifically to double-stranded DNA, using a reference standard of DNA to establish a calibration curve.
Liquid chromatography may also be used to determine the concentration of plasmid DNA using a reference standard. In some cases, capillary electrophoresis is also acceptable.
Residual host-cell DNA
The content of residual host-cell DNA is determined using a suitable method, unless the production process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
Sterility (2.6.1)
It complies with the test for sterility.
Viruses used for production
Their production is based on a seed lot and a cell-bank system or, where applicable (for example, for baculoviruses), on a transient system. The strain of virus used is identified by historical records that include information on its origin and its subsequent manipulation, notably deleted or modified regions. The nucleotide sequence of the viruses is documented.
Only a virus used for production that complies with the following requirements may be used.
Identification
Viruses used for production are identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Genomic integrity
Genomic integrity of the virus used for production is verified by suitable methods such as restriction enzyme analysis. Where viruses are modified to express rep or cap genes or the expression cassette, genomic integrity is assessed by sequencing or by quantitative PCR of these regions.
Genetic stability
Where a stable production system is used, genetic stability is verified at or beyond the maximum number of cell doublings used for production.
Virus titration
The infectious titre is determined by a suitable assay.
Wild-type AAV
Where applicable, the absence of wild-type AAV in helper virus seed lots is verified using NAT (2.6.21).
Replication-competent viruses
Detection of replication-competent viruses is performed by suitable methods. No replication-competent viruses are found.
Extraneous agents (2.6.16)
It complies with the test for extraneous agents. In addition, detection of potential contamination with specific insect viruses is required where applicable.
PRODUCTION AND HARVEST
All processing of the cell bank and subsequent cell cultures is done in an area with a suitable classified space with appropriate containment level where no other cells, viruses or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media is qualified. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production, and at no stage during production is penicillin or streptomycin used. A portion of the production cell cultures is set aside as uninfected cell cultures (control cells).
Each single harvest that complies with the following requirements may be used in the preparation of the purified harvest.
Identification
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Vector concentration
The titre of infectious vector and the concentration of vector particles in single harvests are determined.
Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous agents.
Control cells
Control cells comply with a test for identification (5.2.3) and a test for extraneous agents (2.6.16) and specific insect viruses, where insect cell lines are used for production.
PURIFIED HARVEST
Several single harvests may be pooled before the purification process. The purification process is validated to demonstrate satisfactory removal of impurities.
Purified harvests that comply with the following requirements may be used in the preparation of the final bulk.
Identification
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
Genetic characterisation
The following tests are carried out.
Vector concentration
The titre of infectious vector and the concentration of vector particles are determined.
Residual viruses used for production
Residual viruses used for production are assessed by plaque assays or tissue culture infective dose 50 (TCID50) on permissive cell lines or by quantitative PCR, according to the production system used.
Residual proteins
The concentrations of residual host-cell and/or viral proteins are determined by a suitable immunochemical method (2.7.1), unless the process has been validated to demonstrate suitable clearance.
Residual DNA
The content of residual producer-cell DNA and of residual DNA from intermediates such as plasmids and production viruses where applicable, is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Residual reagents
Where reagents are used during production, tests for these substances are carried out on the purified harvest, unless the process has been validated to demonstrate suitable clearance.
Residual antibiotics
Where antibiotics are used during the production process, their residual concentration is determined by a microbiological assay (adapted from general method 2.7.2) or by other suitable methods (for example, liquid chromatography), unless the process has been validated to demonstrate suitable clearance.
FINAL BULK
Several purified harvests may be pooled during preparation of the final bulk. A stabiliser and other excipients may be added. The formulated product is filtered through a bacteria-retentive filter.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.
Sterility (2.6.1)
It complies with the test for sterility.
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.
Provided that the tests for bovine serum albumin (when bovine serum is used to manufacture the vector), replication-competent AAV and residual viruses used for production have been carried out with satisfactory results on the final bulk, they may be omitted on the final lot.
IDENTIFICATION
The vector is identified by immunochemical methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
TESTS
Osmolality (2.2.35)
Within the limits approved for the particular preparation.
pH (2.2.3)
Within the limits approved for the particular preparation.
Extractable volume (2.9.17)
It complies with the test for extractable volume.
Residual moisture (2.5.12)
Within the limits approved for the particular freeze-dried product.
Bovine serum albumin
Where bovine serum has been used during production, not more than the limit approved for the particular preparation, determined by a suitable immunochemical method (2.7.1).
Replication-competent AAV concentration
Within the limits approved by the competent authority.
Detection of replication-competent AAV is performed by a replication assay on a permissive cell line previously infected with a helper virus and analysis of the replicative forms by Southern blot on low-molecular-weight DNA, or by detection of the rep gene by quantitative PCR.
Vector aggregates
Vector aggregates are determined by suitable methods (for example, light scattering).
Sterility (2.6.1)
It complies with the test for sterility.
Bacterial endotoxins (2.6.14)
Less than the limit approved for the particular preparation.
ASSAY
Vector-particle concentration
Vector-particle concentration is determined using a suitable method such as quantitative PCR by comparison with a standard curve obtained using the recombinant AAV plasmid or an AAV reference standard. This concentration is within the limits approved for the particular product.
Infectious vector titre
Titrate the preparation to be examined by inoculation into cell cultures. Titrate an appropriate vector reference standard to validate each assay.
The infectious vector titre of the preparation to be examined is not less than the minimum amount stated on the label.
The assay is invalid if:
Ratio of vector-particle concentration to infectious vector titre
Within the limits approved for the particular product.
Expression of the genetic insert product
The expression of the genetic insert product is determined wherever possible, following inoculation of cell cultures with the product at a predetermined multiplicity of infection, by suitable immunochemical (2.7.1) or biochemical assays or by flow cytometry (2.7.24).
Biological activity
Unless otherwise justified and authorised, biological activity is determined by a suitable in vitro or in vivo test.